Authors: Meiqi Wang, Lin Feng, Pei Wu, Yang Liu, Hongmei Ren, Xiaowan Jin, Xiaoqiu Zhou, Weidan Jiang
Categories: Original Research Article, Myo-inositol, Growth performance, Hypertrophy, Myonuclear, Protein synthesis, Grass carp
Source: Animal Nutrition
Authors: Meiqi Wang, Lin Feng, Pei Wu, Yang Liu, Hongmei Ren, Xiaowan Jin, Xiaoqiu Zhou, Weidan Jiang
Skeletal muscle constitutes the largest tissue in fish and represents the primary edible portion. Given the substantial differences in muscle growth dynamics between fish and mammals, fish serve as a crucial and distinctive model organism for investigating the fundamental mechanisms of growth regulation in vertebrates. Myo-inositol (MI), originally discovered in muscle, plays significant biological roles in growth regulation, membrane biogenesis, and transmembrane signal transduction. However, to date, no studies have investigated how MI affects adult fish growth and muscle development. A total of 450 adult grass carp (Ctenopharyngodon idella) (704.84 ± 0.91 g) were randomly divided into 6 treatments with 3 replicates of 25 fish each to receive dietary MI at 35.38 (basal diet, deficient group), 98.12, 195.21, 292.30, 389.39, and 486.48 mg/kg. The trial period lasted for 8 weeks. Results indicated that compared with the 35.38 mg/kg MI group, all groups supplemented with MI improved the specific growth rate (SGR) and percent weight gain (PWG) of adult grass carp (P < 0.05). Compared with the deficient group, the sodium-myo-inositol cotransporter 2 (SMIT2) and MI content in the muscle of grass carp in the 292.30 to 486 mg/kg MI group were significantly elevated (P < 0.05). Compared with the deficient group, the dietary MI levels of 195.21 to 486.48 mg/kg lead to increased myofiber mean diameters and the frequency of myofibers with a diameter > 100 μm, while decreased myofiber diameters < 60 μm (P < 0.05). This implies that MI promotes muscle hypertrophy. The hypertrophic effect of MI was primarily ascribed to an increase in the number of myonuclear and enhanced protein synthesis, which is associated with the regulation of the skeletal muscle lysyl oxidase (LOX) and the protein kinase B (AKT)/target of rapamycin (TOR)/ribosomal protein S6 kinase 1 (S6K1) signaling pathways. Additionally, MI inhibited the myostatin (MSTN) and the forkhead box O3 (FoxO3)/muscle RING-finger protein-1 (MuRF1)/muscle atrophy F-box (MAFbx) pathways, which are involved in muscle atrophy and protein degradation. Based on PWG, the appropriate MI requirement of adult grass carp was determined to be 301.30 mg/kg. This study offers a preliminary theoretical foundation for the potential mechanism by which MI promotes muscle hypertrophy in fish and furnishes a reference for the commercial feed formulation of adult grass carp.
Skeletal muscle constitutes the largest tissue in fish (Perez et al., 2023). Fish have more muscle than any other vertebrates, accounting for more than 50% to 60% of the individual body weight (Rossi and Borowiec, 2024). For humans, fish meat provides high-quality protein to the diet (Boyd et al., 2022). The smallest cellular unit constituting skeletal muscle tissue is the multinucleated myofiber (Feng et al., 2024). Growth and plasticity of myofibers in fish are modulated by both extrinsic and intrinsic factors, including nutrition, growth factors, and environment (Mukund and Subramaniam, 2020). Previous studies have demonstrated that vitamin nutrition such as niacin supplement contributes to the growth and improved muscle quality of grass carp (Ctenopharyngodon idella) (Sun et al., 2024). Myo-inositol (MI), a water-soluble vitamin, exists either in its free form or as a component of phospholipids and inositol phosphate derivatives (Croze and Soulage, 2013). MI can be synthesized de novo from glucose in a wide range of animal tissues to meet physiological requirements. However, for certain aquatic animal species, the de novo synthesis of MI is inadequate to satisfy their metabolic demands and requires exogenous addition and acquisition through the action of inositol transporter proteins (Cui et al., 2022). In fish, the kidney is a crucial organ for inositol catabolism. MI is decomposed into D-glucuronic acid under the effect of inositol oxygenase and is further metabolized to participate in the pentose phosphate cycle (Croze and Soulage, 2013). As a crucial nutrient, MI performs significant biological functions in growth regulation, membrane biogenesis, energy homeostasis, and transmembrane signal transduction (Gonzalez-Uarquin et al., 2020; Mai et al., 2001). According to previous experiments, grass carp gut and skin become damaged by MI deficiency (Li et al., 2017; Wang et al., 2024). Furthermore, the previous research performed has shown that Jian carp (Cyprinus carpio var. Jian) suffers skin and muscle lesions due to a deficiency of MI in their diet, and MI has been shown to enhance the antioxidant status of Jian carp muscle (Jiang et al., 2010). Meanwhile, Scherer identified MI in muscle tissue, referring to it as muscle ‘sugar’ (Shirmohammad et al., 2016). Therefore, MI may be indispensable for the growth and development of fish myofibers, and specific mechanisms of action need to be clarified by more comprehensive studies.
Unlike mammals, fish muscle possesses unlimited growth capacity and myofiber development, encompassing hyperplasia and hypertrophy (Perez et al., 2023). Muscle growth is dominated by hypertrophy in adult fish (de Almeida et al., 2008). Myofiber size increases during skeletal muscle hypertrophy, and this occurs through two an increased number of myonuclear and protein synthesis (Fukada and Ito, 2021). Myonuclear numbers and spatial distributions are critical for myofiber function and are determinants of animals' skeletal muscle size (Cramer et al., 2020). It has been discovered that the regulation of myonuclear may be closely associated with the lysyl oxidase (LOX) pathway in mouse myoblasts (Li et al., 1997). However, the effect of MI on the regulation mechanism of the number of myonuclear in animals has not been reported. Divecha and colleagues reviewed other studies and proposed that there are several nuclear processes that may be influenced by MI, including chromatin structure, DNA synthesis and the cell cycle (Cocco et al., 1988; D’Santos et al., 1998). The transcription of LOX in rats is regulated by the antioxidant element (ARE) on the promoter (Li et al., 2011). Previous study discovered that MI can activate NF-E2-related factor 2 (Nrf2) (Jiang et al., 2014, 2015). Meanwhile, Nrf2 can bind to LOX (ARE) to regulate the transcription of the lox gene in rat fibroblasts (Li et al., 2015). It is essential to emphasize that LOX functions as a copper-dependent enzyme, with MI playing a vital role in chelating copper ions to sustain copper homeostasis (Jiang et al., 2013). Furthermore, myofibers have been demonstrated to secrete lysyl oxidase like 3 (LoxL3), which oxidatively modifies fibronectin (FN), thereby activating integrin-mediated signal transduction in mice (Kraft-Sheleg et al., 2016). FN has also been reported to stimulate peripheral nuclear localization in mice by activating integrins (Fukada and Ito, 2021; Roman et al., 2018). MI could potentially regulate the transcription and/or activation of LOX, subsequently influencing the LOX/FN signaling pathway, which is integral to the growth and development of myofibers. Consequently, further research is imperative to substantiate these preliminary observations.
In addition to the increase in the number of myonuclear, nutrients, particularly protein deposition, is another major factor in skeletal muscle hypertrophy (Fukada and Ito, 2021). The balance between protein synthesis and degradation governs protein deposition. The core pathway of protein synthesis is the insulin-like growth factor-1 (IGF-1)/target of rapamycin (TOR)/ribosomal protein S6 kinase 1 (S6K1). Protein degradation is regulated by a pathway involving ubiquitin and proteasomes, and the transcriptional regulator forkhead box O3 (FoxO3) can activate the ubiquitin-proteasome system (Sandri et al., 2004). Currently, there are only two reports in aquatic animals investigating the impact of MI on protein deposition in muscle (Bu et al., 2022; Lu et al., 2023). The synthesis product of MI, phosphatidylinositol (PI), is converted into phosphatidylinositol 3-phosphate (PtdIns3P) under the catalysis of phosphatidylinositol 3-kinase (PI3K), and PI3K is closely linked to the protein synthesis pathway. Consequently, the mechanisms by which MI influences skeletal muscle protein synthesis and degradation require further detailed investigation.
The hypertrophy of the skeletal muscles can be induced by a variety of factors (Bamman et al., 2018). In addition to the increase in myonuclear and protein synthesis, muscle hypertrophy is also regulated by the negative regulator myostatin (MSTN) and the positive myogenic regulatory factors (MRFs) (such as myogenin [MyoG] and myogenic determining [MyoD]) (Feng et al., 2024). Follistatin (FST) is mainly involved in inhibiting MSTN and regulating muscle growth (Winbanks et al., 2012). Nevertheless, the process by which MI regulates MSTN signaling and MRFs to stimulate skeletal muscle hypertrophy in fish remains unclear. It has been reported that MI has insulin-like activity, which might be attributed to the role of inositol phosphoglycans (IPG) as the second messenger of insulin (Croze and Soulage, 2013; Kim et al., 2014). A study has demonstrated that insulin can release activin A in macrophages in humans (Cuschieri et al., 2008). Furthermore, activin A can induce the expression of fst mRNA in different pituitary cell types of grass carp (Fung et al., 2017). In a previous study, it was found that appropriate dietary MI could increase the transcription of IGF-1 in the liver of hybrid grouper (Epinephelus. fuscoguttatus♀ × E. lanceolatus♂) (Tian et al., 2024). In mouse C2C12 myoblasts, IGF-1 has been reported to prevent MSTN inhibition (Yang et al., 2007). Meanwhile, IGF-1 can induce the expression or activity of MRFs to promote muscle differentiation (Musaro and Rosenthal, 1999). Therefore, MI may influence the development of myofibers and is associated with the FST/MSTN/MRFs pathway.
Grass carp is a highly significant species in freshwater aquaculture, possessing considerable economic value (Wu et al., 2012). It is commonly held that grass carp grow in three distinct juvenile, young (on-growing), and adult (Li et al., 2010). Based on previous studies, the MI requirements of juvenile grass carp are lower than those of young grass carp. Specifically, the MI requirement for juvenile grass carp has been determined to be 166.0 mg/kg. Previous investigations revealed that the MI requirement for grass carp is 276.7 mg/kg during on-growing (Li et al., 2017). However, current research on the dietary MI requirements of adult grass carp is scarce, and require further studies.
The purpose of this study was to investigate the influence of MI on the growth performance, growth and development of myofibers, as well as the number of myonuclear in adult grass carp for the first time. The main objectives are as the potential mechanisms underlying MI-induced muscle hypertrophy in grass carp. Meanwhile, the MI requirements of adult grass carp as a reference for the commercialization of fish compound feeds. Dietary MI levels might promote muscle hypertrophy in adult grass carp by increasing the number of myonuclear, enhancing muscle protein synthesis, and inhibiting the MSTN and the FoxO3/muscle RING-finger protein-1(MuRF1)/muscle atrophy F-box (MAFbx) pathways.
According to the Chinese ethical guidelines for experimental animals, the Animal Care Advisory Committee of Sichuan Agricultural University approved all animal studies (No. WMQ-2022114015).
The composition and nutrient levels of the basal diet is provided in Table 1. Dietary protein was mainly sourced from fish meal, casein, and gelatin, and dietary lipid was derived from fish oil and soybean oil. MI was purchased from Sichuan Bohanda Biotechnology (Zigong, China). The purity of MI was 96.50%. Using MI premix as a supplement to a basal diet, six levels were basal diet (without supplement), 100, 200, 300, 400, and 500 mg MI/kg diets. The ingredients in all diets were mixed evenly and made into granules, dried in an oven, and refrigerated at 4 °C. In six experimental diets, the MI levels of 35.38 (basal diet), 98.12, 195.21, 292.30, 389.39, and 486.48 mg/kg diet were determined with reference to Frieler (Frieler et al., 2009). In this method, aqueous extract and benzoyl chloride were used as modifiers in a precolumn derivatization reaction. Through reverse-phase gradient chromatography using HPLC, the benzoylated derivatives were separated, and their absorbance was detected at 231 nm for quantification.Table 1Composition and nutrient levels of the basal diet (air-dry basis, g/kg).Table 1IngredientsContentNutrient levels5ContentFish meal (CP, 66.25%)100.00Crude protein267.24Casein (CP, 75.87%)170.00Crude lipid41.81Gelatin (CP, 82.68%)73.98ω-3 PUFA9.95Fish oil20.20ω-6 PUFA9.65Soybean oil18.20Available phosphorus4.00Corn starch188.38Organic matter853.50a-Starch300.00Gross energy, MJ/kg17.57Microcrystalline cellulose65.00CaH2PO411.30Mineral premix120.00Vitamin premix (myo-inositol free)210.00Choline chloride310.00Myo-inositol premix410.00Butylated hydroxyanisole0.15L-Thr (97.5%)0.91L-Trp (98%)1.88Total1000.00CP = crude protein; PUFA = polyunsaturated fatty acids.1Per kilogram of mineral premix (g/kg): MnSO4·H2O (31.8% Mn), 2.66; MgSO4·H2O (15.0% Mg), 256.79; FeSO4·H2O (30.0% Fe), 12.61; ZnSO4·H2O (34.5% Zn), 8.87; CuSO4·5H2O (25.0% Cu), 0.95; Ca (IO3)2 (3.2% I), 1.56; selenium yeast (0.2% Se) 13.65. All ingredients were diluted with corn starch to 1 kg.2Per kilogram of vitamin premix (g/kg):VA (500,000 IU/g), 0.44; VD3 (500,000 IU/g), 0.19; VE (50%), 25.50; VK3 (50%), 0.38; VB12 (1%), 0.94; D-biotin (2%), 1.05; folic acid (95%), 0.17; thiamin nitrate (98%), 0.11; VC (95%), 9.77; nicotinic acid (99%), 3.44; calcium pantothenate (93.1%), 4.42; riboflavin (80%), 0.73; VB6 (98%), 0.55. All ingredients were diluted with corn starch to 1 kg.3Each kilogram of choline chloride product contains 306.71 g of choline chloride with a purity of 50%. The remaining part is diluted to 1 kg using corn starch.4For treatments 1 to 6, the amounts of myo-inositol, supplementation are 0.000, 0.067, 0.171, 0.274, 0.378, and 0.481 g, respectively, and the remainder is filled with microcrystalline cellulose to make up 1 kg.5Available phosphorus, ω-3 PUFA, and ω-6 PUFA were calculated according to NRC (2011).
In breeding barrels with a diameter of 1 m and height of 1 m, 450 healthy grass carp were maintained. After purchase, the environment was acclimatized for four weeks. In the pre-feeding period, adult grass carp (704.84 ± 0.91 g) were randomly distributed among 6 groups, with three replications each consisting of 25 fish. Six groups of grass carp were fed a basal diet for 14 days without additional MI supplementation, according to the previous study (Li et al., 2017). During the trial period, 4 meals (07:00, 00, 00, and 00) were provided per day for 8 weeks, and no grass carp died. The temperature, dissolved oxygen levels, and pH were monitored daily during the trial. A temperature range of 26.0 to 30.7 °C, oxygen concentrations of > 6.0 mg/L and pH levels of 7.0 to 7.5 were measured.
The crude lipid and crude protein contents of dried samples and muscle tissue were determined by the Soxhlet (method 2003.05) and Kjeldahl methods (method 990.03) according to the AOAC standards (AOAC, 2005). The dry matter content was determined by drying the samples at 105 °C on an oven for 4 h (method 930.15) (AOAC, 2005). Standard procedures of the Association of Official Analytical Chemists were used to determine crude ash (method 942.05) (AOAC, 2005). The organic matter (OM) content of the feeds was calculated by dry matter subtracting crude ash content. The gross energy was accurately determined using an oxygen bomb calorimeter (model 6400, Parr Instrument Company, Moline, IL, USA) following the standard method of ISO 1998 (ISO, 1998).
A 24-h fast was imposed on all fish upon the conclusion of the experiment. Subsequently, growth performance was measured by weighing the fish in each experimental breeding barrel. After weighing, 12 fish were randomly selected from each treatment group and anesthetized with benzocaine bath (50 mg/L) and euthanized by a cranial blow (Xiao et al., 2023). Biochemical analysis was conducted after muscle samples were frozen in liquid nitrogen and stored at −80 °C. In each group, three grass carp were randomly chosen for muscle collection, which was then fixed in a 4% paraformaldehyde and subjected to histological examination. Hematoxylin-eosin (H&E) staining was used to stain 4 μm paraffin sections. ImageJ software was used to measure the myofiber mean diameters of three images (for each picture, 200 myofibers were chosen at random). The frequency of various myofiber sizes (<60, 60–100, and >100 μm) was measured employing the method of Tian et al. (2023). The three key indicators in the muscle tissue of grass carp, including section area, number of fibers, and total fiber count, were statistically measured using Yu et al. (2024) method.
In each growth stage, after sampling, a GD fixation solution (G1111, Servicebio, Wuhan, Hubei, China) was employed to preserve the muscle tissue as well as embed it in paraffin. In the next step, 5 μm section of the tissue was cut, dewaxed with xylene, and stained with H&E after rehydration. IF staining was also carried out using dewaxed slides, which were blocked with a blocking buffer after treatment with an antigen repair solution. Then the slides were incubated with primary antibody at 4 °C for 17 h followed by 2 h incubation in the dark with fluorescent secondary antibody, and 4′,6-diamidino-2-phenylindole (DAPI) staining (C1006, Beyotime, Shanghai, China) for 10 min. Finally, the anti-fading fluorescent sealant (Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China) was utilized to seal the slide and cover the cover glass. Images were captured using an inverted fluorescence microscope (DMI4000B, Leica, Germany) and quantified using Image-Pro-Plus (version 5.0, Media Cybernetics Inc., Silver Spring, MD, USA). The primary antibody information of immunofluorescence is presented in Table S1.
As per the instructions, Na^+^/K^+^-ATPase activity in the intestine was analyzed using the microplate method (No. A070-2-2, Nanjing Jiancheng Institute of Bioengineering, Nanjing, Jiangsu, China). With the kit, the concentration of proteins was analyzed (No. A045–3, Nanjing Jiancheng Institute of Bioengineering, Nanjing, Jiangsu, China). An ELISA kit was utilized to analyze liver and muscle samples for MI content (No. CB10576-Fi, Shanghai COIBO BIO, Shanghai, China).
Assays were carried out using Trizol (No. RR047A, TaKaRa, Dalian, Liaoning, China) to prepare total RNA extracts. Nucleic acid electrophoresis was employed to ensure the quality of RNA. Subsequently, a cDNA synthesis kit was employed to synthesize the cDNA (No. R123–01, Vazyme, Nanjing, Jiangsu, China). The RT-PCR was performed in the Quant-Studio 5 Flex system (QuantStudio 5384 holes, Applied Biosystems, CA, USA), and the final reaction volume was 10 μL with the use of the Fast SYBR Green qPCR Master Mix kit (No. PC3301, Aidlab, Beijing, China). The RT-PCR cycling condition were as 95 °C for 30 s (hold stage), followed by 95 °C for 15 s and 60 °C for 34 s (measure fluorescence), finally 60 °C for 1 min and 95 °C for 15 s for 40 cycles (melt curve stage). Quantification of gene expression was conducted using the 2^−ΔΔCt^ method. β-Actin was used as an internal reference. In Table S2, the primer sequences are provided.
The radio immunoprecipitation assays (RIPA) (No. P0013B, Beyotime, Shanghai, China) on muscle samples to extract total protein. With the kit, nuclear proteins were extracted (No. P0027, Beyotime, Shanghai, China). The protein concentration of each sample was analyzed using protein detection kits of BCA (No. A045-4-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). After sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel separation, protein samples were transferred to polyvinylidene fluoride (PVDF) membranes, and overnight incubation with primary antibodies was carried out at 4 °C. The incubation of the primary antibody was followed by the addition of the secondary antibody to the PVDF membrane. With an enhanced chemiluminescence system (ECL), the bands were visualized, and the results were analyzed using Image J. Information about antibodies is provided in Table S3.
In Table S4, formulas are provided for calculating growth performance parameters.
This experiment followed a completely random design. The data was analyzed using SPSS 27.0 (SPSS Inc., Chicago, IL, USA) using one-way analyses of variance (ANOVA) and Duncan’s multiple comparisons. According to the statistical model,Yij=μ+Ti+εij,where Yij is the observation of dependent variables; μ is the overall mean; Ti is the group difference; and εij is the residual error for the observation. The data is presented as mean and standard error of the mean (SEM). P < 0.05 was considered to be statistically significant. A quadratic regression model was used to calculate the dietary MI requirements based on the results of R^2^ and P-value using linear and quadratic regressions.
An assessment of the growth performance and nutrient composition of muscles under different levels of MI supplementation was performed (Table 2). Compared with the 35.38 mg/kg MI group (deficient group), supplementation of MI showed a significant increase in the final body weight (FBW), percent weight gain (PWG), specific growth rate (SGR), feed intake (FI), and feed efficiency (FE), as well as whole length, body length, width, and height (P < 0.05). Grass carp fed diet added with 195.21 to 389.39 mg/kg MI had a significantly higher condition factor (CF) than a deficiency diet (P = 0.006). However, all MI supplements did not significantly differ in the visceral index (VSI) and hepatosomatic index (HSI) (P > 0.05). There were significantly reduced moisture levels on grass carp fed diets that contained 195.21 to 486.48 mg/kg MI in comparison to those fed MI deficient diets (P = 0.012). At 292.30 mg/kg MI level, improvements in crude lipid and crude protein were observed (P < 0.05). According to the quadratic regression analysis of PWG, adult grass carp required 301.30 mg/kg MI in their diets (Y = −0.0005X^2^ + 0.3013X + 49.874, R^2^ = 0.8467).Table 2Effects of diets containing graded levels of MI on the growth performance and nutrient composition in the muscle of adult grass carp (Ctenopharyngodon idella).Table 2ItemDietary MI levels, mg/kg dietSEMP-value35.3898.12195.21292.30389.39486.48ANOVALinearQuadraticIBW1, g/fish705.80703.50705.10704.00705.00705.620.3910.3140.8110.249FBW1, g/fish1008.03^a^1295.47^bc^1318.68^c^1331.69^c^1330.13^c^1218.53^b^23.985<0.0010.007<0.001PWG1, %54.17^a^84.01^bc^86.78^c^89.34^c^88.76^c^72.73^b^3.415<0.0010.006<0.001SGR1, %/d0.77^a^1.09^bc^1.11^c^1.14^c^1.13^c^0.97^b^0.036<0.0010.005<0.001FI1, g/fish1065.76^a^1215.97^bc^1220.27^c^1220.69^c^1221.17^c^1205.98^b^13.759<0.001<0.001<0.001FE10.36^a^0.49^bc^0.50^bc^0.51^c^0.51^c^0.43^ab^0.0160.0030.057<0.001Whole length2, cm43.42^a^45.67^b^48.25^d^47.50^cd^47.58^cd^46.50^bc^0.319<0.001<0.001<0.001Body length2, cm37.00^a^39.17^b^41.50^d^40.75^cd^40.75^cd^39.67^bc^0.290<0.001<0.001<0.001Body width2, cm5.15^a^5.42^b^5.50^bc^5.75^c^5.73^c^5.58^bc^0.049<0.001<0.001<0.006Body height2, cm8.00^a^8.70^b^9.13^c^9.25^c^9.28^c^9.05^c^0.086<0.001<0.001<0.006CF2, g/cm^3^1.84^a^1.88^a^1.99^b^2.00^b^2.02^b^1.92^ab^0.0170.0060.0120.003VSI2, %10.4310.6511.3311.2011.0711.040.1930.7860.3250.327HSI2, %2.792.812.903.122.942.910.0450.3340.1920.192**Muscle composition, %**Moisture279.15^b^78.36^ab^77.88^a^77.46^a^77.67^a^78.05^a^0.1530.0120.0060.006Crude protein217.37^a^18.67^b^19.63^bc^20.12^c^19.71^bc^18.67^b^0.209<0.0010.003<0.001Crude lipid21.74^a^1.89^a^2.24^bc^2.41^c^2.18^b^2.14^b^0.045<0.001<0.001<0.001MI = myo-inositol; IBW = initial body weight; FBW = final body weight; FI = feed intake; FE = feed efficiency; PWG = percent weight gain; SGR = specific growth rate; CF = condition factor; VSI = viscerosomatic index; HSI = hepatosomatic index.Mean values within a row with different superscript letters indicate significant difference (P < 0.05).1n = 3 (for 3 replicate groups, 25 fish per replicate).2n = 3 (for 3 replicate groups, 2 fish per replicate).
The MI uptake and transport are dependent on the sodium gradient. The MI contents and Na^+^/K^+^-ATPase in the organizations are presented in Table 3. The sodium-myo-inositol cotransporter (SMIT1 and SMIT2) are shown in Fig. 1B and C. Results showed that the MI contents and SMIT2 protein levels in the muscle increased significantly from 292.30 to 486.48 mg/kg MI in comparison to the MI deficient group (P < 0.05). As presented in Table 3, there was a significant increase of Na^+^/K^+^-ATPase activity in the intestine when the MI level was increased compared to a deficient group (P < 0.001). However, in terms of SMIT1 protein levels, MI supplement groups did not differ significantly from each other (P > 0.05).Table 3Effects of diets containing graded levels of MI on the MI contents and enzymatic activity of tissues of adult grass carp (Ctenopharyngodon idella).Table 3ItemDietary MI levels, mg/kg dietSEMP-value35.3898.12195.21292.30389.39486.48ANOVALinearQuadraticLiver MI content, mg/g prot0.73^a^0.82^a^0.98^b^1.19^c^1.26^c^1.37^d^0.041<0.001<0.0010.394Muscle MI content, mg/g prot0.78^a^0.85^ab^0.85^ab^0.90^b^0.90^b^0.91^b^0.0120.004<0.0010.261Intestine Na^+^/K^+^-ATPase, U/mg tissue01.09^a^01.34^b^1.66^c^1.67^c^1.77^c^1.96^d^0.052<0.001<0.0010.011MI = myo-inositol; Na^+;^/K^+^-ATPase = Na^+^/K^+^-adenosine-triphosphate.Mean values within a row with different superscript letters indicate significant difference (P < 0.05). (n = 6).Fig. 1Effects of dietary myo-inositol (MI) levels on muscle histological and MI transport. (A) Cross-sectional microstructure (×200, scale bar = 50 μm) of grass carp muscle; (B, C) SMIT1 and SMIT2 protein levels in muscle. Results were expressed as mean and SEM (n = 6). The columns are marked with different letters to indicate significant differences (P < 0.05). SMIT1 = sodium-myo-inositol cotransporter 1; SMIT2 = sodium-myo-inositol cotransporter 2; GAPDH = glyceraldehyde-3-phosphate dehydrogenase.Fig. 1
To determine whether supplementing grass carp with MI affects the growth and development of their myofibers, the frequency distribution of myofiber diameters at different developmental stages was analyzed. As shown in Fig. S1, at the juvenile grass carp stage, <60 μm and 60 to 100 μm myofiber diameter frequencies were significantly higher than >100 μm frequencies (P < 0.001). During the young grass carp stage, the myofiber diameters were the highest at 60 to 100 μm frequencies. At the adult grass carp stage, 60 to 100 μm and >100 μm myofiber diameter frequencies were significantly higher than <60 μm frequencies (P = 0.007).
The adult grass carp’s myofiber characteristics are influenced by MI and the results are presented in Table 4. In supplemental MI groups, myofiber mean diameters were significantly larger than in the MI-deficient group (P < 0.001), and myofiber diameter >100 μm frequencies were also higher (P = 0.002). The <60 μm diameter frequencies (195.21 and 292.30 mg MI/kg) were markedly reduced compared to those of the MI-deficient group (P < 0.001); the diameters of 60 to 100 μm frequencies showed an opposite trend (P < 0.05). The section area was significantly reduced in the MI-deficient group (P < 0.001), and the other groups had significantly decreased number of fibers and decreased total number of myofibers (P < 0.05).Table 4Effects of diets with different MI levels on the muscle histology of grass carp (Ctenopharyngodon idella).Table 4ItemDietary MI levels, mg/kg dietSEMP-value35.3898.12195.21292.30389.39486.48ANOVALinearQuadraticMean diameter, μm69.56^a^77.24^b^81.88^bc^87.67^d^88.91^d^84.56^cd^1.716<0.001<0.001<0.001Frequency of myofibers (<60 µm)47.56^c^43.33^c^34.22^b^21.56^a^28.33^ab^33.11^b^2.318<0.001<0.0010.001Frequency of myofibers (60–100 µm)28.89^a^32.00^ab^42.22^c^42.22^c^37.67^bc^33.19^ab^1.5120.0130.0970.001Frequency of myofibers (>100 µm)21.42^a^27.78^b^33.78^bc^36.22^c^34.00^bc^31.56^bc^1.3640.002<0.001<0.001Section area, cm^2^30.45^a^37.16^b^39.98^b^41.37^b^41.41^b^38.30^b^1.023<0.001<0.001<0.001Number of fibers1, ×10^4^ cm^2^2.65^c^2.14^b^1.90^ab^1.67^a^1.61^a^1.78^a^0.091<0.001<0.001<0.001Total number of fibers2, ×10^5^ cm^2^8.06^b^7.94^b^7.60^ab^6.90^a^6.68^a^6.82^a^0.1720.0280.0020.553MI = myo-inositol.Mean values within a row with different superscript letters indicate significant difference (P < 0.05). (n = 3).1The density of myofibers.2Estimated from total area and number of myofibers per square centimeter.
To assess whether MI promotes muscle hypertrophy, the number of myonuclear and other indicators that promote hypertrophy were measured (Fig. 2, Fig. 3). In Fig. 2A and B, other groups had significantly higher numbers of myonuclear than the MI-deficient group (P < 0.001). As shown in Fig. 2D–H, diet supplemented with 292.30 mg/kg MI had significantly increased LOX, fibronectin and integrin α5 protein levels in grass carp than those fed the deficient diet (P < 0.05). However, none of the MI supplements significantly affected integrin α7 protein levels (P > 0.05). The immunofluorescence expression level of LOX, integrin α5, and fibronectin and found similar results to protein levels (Fig. 3). As shown in Fig. 2C–E, adult grass carp fed diets added with 292.30 to 468.48 mg/kg MI had higher nrf2 mRNA and protein levels than those fed the diet deficient in MI (P = 0.008).Fig. 2Effects of dietary myo-inositol (MI) levels on the number of myonuclear and the index of promoting hypertrophy. (A) The number of myonuclear in muscle tissue by 4′,6-diamidino-2-phenylindole (DAPI) staining (×200, scale bar = 50 μm). (B) The quantitative analysis result of the number of myonuclear in (A) (n = 3). (C) The mRNA level of nrf2 in muscle (n = 6). (D–H) The proteins levels of Nrf2 and LOX/fibronectin/integrin pathway in grass carp muscle (n = 6). Results were expressed as mean and SEM, indicating significant differences with different letters (P < 0.05).Fig. 2Fig. 3Effects of dietary myo-inositol (MI) levels on integrin α5, LOX, fibronectin protein fluorescence intensity. (A, B) The immunofluorescence levels of integrin α5 in grass carp muscle (n = 3). (C, D) The immunofluorescence levels of LOX in grass carp muscle (n = 3). (E, F) The immunofluorescence levels of fibronectin in grass carp muscle (n = 3). Data are presented as mean and SEM and one-way ANOVA was performed. DAPI = 4′,6-diamidino-2′-phenylindole.Fig. 3
The FST protein levels significantly increased and plateaued after MI level reached 195.21 mg/kg (P < 0.067). A grass carp-fed diet added with 98.12 to 389.39 mg/kg MI showed significantly lower MSTN protein levels than a group that was MI-deficient (P = 0.005). The fluorescence intensity of FST and found similar results with protein expression (Fig. 4C and D).Fig. 4Effects of dietary myo-inositol (MI) levels on myostatin (MSTN)pathway and myogenic regulatory factors. (A, B) The protein levels of LOX/fibronectin/integrin pathway in grass carp muscle (n = 6). (C, D) The immunofluorescence levels of follistatin in grass carp muscle (n = 3). (E, F) Proliferation and differentiation-related genes in myoblasts. (G, H) Protein levels of genes associated with myoblast proliferation and differentiation (n = 6). Results were expressed as mean and SEM, indicating significant differences with different letters (P < 0.05). ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. DAPI = 4′,6-diamidino-2′-phenylindole.Fig. 4
Adult grass carp fed 195.21 and 292.30 mg/kg MI had higher levels of myod, myf5, mrf4, and myhc mRNA, and MyoD and MyoG protein in their muscle than those fed a MI deficient diet (P < 0.05). In the 389.39 mg/kg MI diet, cyclin b and cyclin d mRNA expressions were higher than in the MI-deficient diet (P < 0.05). It was found that mstn and myog mRNA expressions were lower in adult grass carp diets added with 389.39 and 292.30 mg/kg MI, respectively, than in the MI deficient group (P < 0.05). However, the pcna and cyclin e mRNA expression did not differ significantly between the MI supplement groups (P > 0.05) (Fig. 4E and F).
Fig. 5A–H shows muscle protein synthesis and degradation. There were higher levels of igf1, akt, and tor mRNA and p-AKT^Ser473^, p-S6K1^T421/S424^ and p-TOR^Ser2448^ protein in grass carp fed diets supplemented with 195.21 and 292.30 mg/kg MI than those fed a diet deficient in MI (P < 0.05). In the MI-deficient group, pi3k mRNA expression was lower than that in the 486.48 mg/kg MI group (P = 0.029). AKT, S6K1, TOR, and FoxO3a protein levels were unaffected by the MI supplement (P > 0.05). As shown in Fig. 5F–H, the protein level of p-FoxO3a^S253^ at 195.21 mg/kg MI was significantly increased compared to the MI-deficient group (P = 0.003); the MAFbx and MuRF1 protein levels showed an opposite trend (P < 0.05).Fig. 5Effects of dietary myo-inositol (MI) levels on muscle protein deposition in grass carp. (A) The mRNA levels of protein deposition-related genes. (B–E) The protein levels of protein synthesis in grass carp muscle. (F–H) The protein levels of protein degradation in grass carp muscle. Results were expressed as mean and SEM (n = 6), indicating significant differences with different letters (P < 0.05).Fig. 5
As an essential nutrient, MI plays a vital biological role in aquatic animals. In this study, dietary MI is conducive to growth performance (PWG, FI, and FE), somatic parameters (body length, width, height, and CF), and nutritional composition of muscle tissue (crude lipid and crude protein). These results are in line with previous findings on young grass carp (Li et al., 2017). The majority of MI is present in the free form in cells and tissues of animals (Schneider, 2015). The liver is an important tissue that regulates exogenous detoxification and metabolism in the body (Mega et al., 2021). This experiment findings indicate that dietary MI significantly promoted the deposition of MI in the liver tissues. In key tissues, increase in MI content may be related to Na^+^/K^+^-ATPase. Almost all (99.8%) of the free MI intake is absorbed thfrom the intestine via an active transport system involving Na^+^/K^+^-ATPase (Bizzarri and Carlomagno, 2014). Free MI is transported in the intestinal epithelial cells and then to the corresponding target cells in the tissue. This study also revealed that the Na^+^/K^+^-ATPase activity in the intestine tissue was increased by MI. The above results demonstrate that dietary MI may enhance the accumulation of MI in tissues via its interaction with the Na^+^/K^+^-ATPase-driven active transport system. MI regulation of Na^+^/K^+^-ATPase may be related to its function. Na^+^/K^+^-ATPase constitutes a principal component of the sodium-potassium pump, which is integral to the cell membrane. Additionally, phosphatidylinositol, a derivative of MI, is also a constituent of the membrane. Consequently, Na^+^/K^+^-ATPase activity may be indirectly influenced by MI on the targeted membrane.
Most of the free inositol absorbed from the intestinal epithelial cells circulates in the blood and is distributed to various tissues via inositol transporters (Lee and Bedford, 2016). Inositol transporters play a significant role in MI uptake and intracellular distribution (Schneider, 2015). Inositol transporter proteins belong to the solute carrier 5 (SLC5) and are divided into two SMIT1 and SMIT2 and H/myo-inositol transporter (HMIT), of which SLC5A3-encoding SMIT1 and SLC5A11-encoding SMIT2 are expressed in skeletal muscle (Schneider, 2015). This study observed that the protein levels of SMIT2 in adult grass carp were increased by MI addition from 292.30 to 486.48 mg/kg. In this study found that MI deposition in muscle tissues was significantly enhanced by dietary MI, indicating that MI can enter the muscle through the transporter and thereby exert its important functions. Therefore, the following section will discuss the growth and development of fish skeletal muscle in response to MI.
The skeletal muscle of fish is an edible part. It can grow through two hypertrophy (increase in myofiber diameter) and hyperplasia (increase in myofiber number). The myofiber diameter was classified into three stages (<60 μm, 60–100 μm, and >100 μm) (Tian et al., 2023). In grass carp, a myofiber frequency <60 μm was defined as proliferation, and a myofiber frequency >100 μm was defined as hypertrophy. These results in the three stages of juvenile, young, and adult grass carp confirmed that proliferation is more vigorous in the juvenile stage, and muscle hypertrophy is more intense in the adult stage.
It has been reported that when the fish attains approximately 44% of its final body size, the increase of new myofibers will cease during muscle growth, and the increase in muscle mass is mainly through hypertrophy (Zimmerman and Lowery, 1999). Next, it was further demonstrated that the frequency of myofibers with a diameter of <60 μm is decreased in adult grass carp by appropriate MI, and increased diameters of myofibers and frequencies of myofibers with diameters >100 μm have been observed. At the same time, it was also discovered that the area of grass carp segments was augmented by MI, indicating that appropriate dietary MI facilitated myofiber hypertrophy in fish.
Skeletal myofiber is a syncytium containing hundreds of myonuclear (Cisterna and Malatesta, 2024). There is a finite volume of cytoplasm within each nucleus that governs gene transcription and protein synthesis (Ross et al., 2018). In animals, the number and distribution of myonuclear are critical for myofiber function and determinants of skeletal muscle size (Cramer et al., 2020). Substantial studies have indicated that the number of myonuclear increases during muscle hypertrophy (Cramer et al., 2020; Egner et al., 2016). In this study, dietary MI supplementation elevated the number of myonuclear in adult grass carp, suggesting that MI-promoted muscle hypertrophy might be associated with the increase in the number of myonuclear. There is evidence from previous studies that the inositol signaling pathway has multiple nuclear functions. Phosphatidylinositol phosphate lipids (PIPs) regulate nuclear functions, including transcription as well as pre-mRNA splicing and processing (Martelli et al., 2004). In addition, inositol pyrophosphate synthase (IPS) proteins and inositol polyphosphate kinase (IPK) are partially located in the nucleus, and their functions involve the maintenance of telomeres and the export of mRNAs, as well as the repair of DNA (York, 2006). The mechanisms by which MI may affect the number of myonuclear are discussed below.
Integrins constitute a large class of transmembrane adhesion molecules capable of binding to extracellular and intracellular receptors, thereby regulating cell adhesion, migration, and proliferation (Calderwood, 2011). It has been reported that integrin cytoplasmic domain-associated protein-1 (ICAP-1) is capable of transmitting information from integrin-dependent cell adhesion sites to the nucleus to control cell proliferation and gene expression (Fournier et al., 2005). The functions of α5β1 integrin include migration, signal transduction, and growth factor receptor transport (Schaffner et al., 2013). This study observed that MI increased integrin α5 protein levels at an appropriate level, and similar results were noted in immunofluorescence, indicating that MI regulation of integrins may affect nuclear migration, and it is speculated that FN may have been involved in this outcome. FN is a ligand for integrin receptors (Brown et al., 2015). A test in mouse muscle demonstrated that FN enhanced the strength of mouse myofibers through integrin α5β1 (Wu et al., 2011). This study indicated that 292.30 mg/kg MI significantly increased FN protein levels and fluorescence intensity. Co-localization of FN and LOX was detected in rat fibroblast cultures and human tissues (Fogelgren et al., 2005). In addition, LOX can oxidize FN, a modification that enhances FN-induced integrin-mediated signal transduction (Kraft-Sheleg et al., 2016). Therefore, MI may regulate the transcription and/or activation of LOX, thereby influencing the LOX/FN signaling pathway, which is an indispensable component of myofiber growth and development. LOX performs a series of biological functions and is essential for the stability of the cell nucleus. Confocal microscopy has revealed that LOX is present in the nucleus of fibroblasts and plays a functional role in gene regulation through chromatin organization (Li et al., 1997). In this study, it was found that the supplementation of 292.30 mg/kg MI increased LOX protein levels and fluorescence intensity. The increase in protein level of LOX may be related to the activation of Nrf2. The lox gene promoter has an antioxidant element (ARE), and Nrf2 can bind to LOX ARE to regulate lox gene transcription nuclear translocation in rat fibroblasts (Li et al., 2015). In addition, this study found that appropriate MI can increase grass carp muscle nrf2 mRNA and protein levels. Therefore, it is postulated that MI activates Nrf2 and thus may increase LOX protein levels in muscle. Skeletal muscle mainly expresses integrin α7β1, which plays a crucial role in promoting muscle hypertrophy (Boppart and Mahmassani, 2019). Interestingly, the protein level of integrin α7 in muscle was not affected by MI in this study. The possible reason is that there is a negative regulation between integrins α7 and α5 by reducing ligand binding affinity and surface expression (Tomatis et al., 1999).
In addition to the influence of the number of myonuclear on muscle hypertrophy, certain hormones also exert a critical role in muscle mass (Bamman et al., 2018). The transforming growth factor-β (TGF-β) superfamily member MSTN is an effective negative muscle growth regulator (Rodriguez et al., 2014). The results of this study suggested that dietary MI levels of 98.12 to 389.39 mg/kg decreased the level of MSTN protein. The effect of MI on MSTN may be related to FST. It has been reported that FST is a hypertrophic signal and an inhibitor of the negative regulator of muscle MSTN (Lee and McPherron, 2001). The level of FST protein and discovered that appropriate MI enhanced the FST protein levels and fluorescence intensity in the muscle of grass carp. It has been shown that mstn expression in myoblasts is stimulated by IGF-1. It is reported that IGF-1 regulates MSTN transcription in a PI3K/inositol triphosphate 3 (IP3)/calcium-dependent manner by activating the nuclear factor of activated T cells (NFAT) transcription factors (Valdés et al., 2013). Therefore, MI may affect muscle mass and induce muscle hypertrophy through the FST/MSTN/MRF and IGF-1/PI3K/MSTN pathways.
On the other hand, the signal transduction of MSTN plays a crucial role in inhibiting myoblast proliferation and the genes related to terminal differentiation (Rescan, 2001). The proliferation and differentiation of myoblasts are related to PCNA, cyclin and MRFs. The family of MRFs encompasses MyoD and Myf5, which are associated with the proliferation of myofibroblasts, and MyoG and MRF4, which are associated with myofibroblast differentiation and hypertrophy (Johansen and Overturf, 2005). Among myogenic cells, late differentiation is characterized by the protein MyHC (Martelli et al., 2004). According to this study, appropriate MI levels increased cyclin b, cyclin d, myod, mrf4, myf5 and myhc mRNA expression, as well as MyoG and MyoD protein levels. In addition to the effect of MSTN, the up-regulation of MRF by MI may also be related to IGF-1. mrf4 and myog mRNA and protein levels were significantly decreased in the muscles of mice knocked out for IGF-1 (Miyake et al., 2007). This result demonstrated that the addition of MI ranging from 98.12 to 292.30 mg/kg increased mRNA expression of igf1, which proved the hypothesis of this study. The above results indicate that appropriate MI levels promote muscle hypertrophy in fish, which may be affected by myoblast proliferation and differentiation.
The size of fish myofiber depends on the balance between protein synthesis and degradation (Fuentes et al., 2013a, Fuentes et al., 2013b). In simple terms, muscle hypertrophy occurs when protein synthesis exceeds protein degradation; conversely, muscle atrophy occurs. In this study, MI upregulated tor and s6k1 mRNA expression and phosphorylated TOR and S6K1 protein levels. It is likely that dietary MI activates the TOR pathway associated with the IGF-1/PI3K/AKT pathway. There is abundant evidence that shows that the IGF-1/PI3K-AKT/TOR/S6K1 pathway is the central pathway involved in protein synthesis (Fuentes et al., 2013a, Fuentes et al., 2013b). This study observed that MI increased muscle mRNA expression of igf-1, pi3k and akt and protein levels of phosphorylated AKT, indicating that MI promotes protein synthesis and is linked to the IGF-1/PI3K-AKT/TOR/S6K1 pathway.
In eukaryotes, the ubiquitin-proteasome system is of crucial importance for protein degradation (Kitajima et al., 2020). It is a muscle-specific E3 ubiquitin ligase that is MAFbx and MuRF1. These results found that MI reduced MAFbx and MuRF1 protein levels, which may be related to FoxO. It is reported that the nuclear localization of AKT-mediated FoxO transcription factors is essential for the activation of transcription for MuRF1 and MAFbx (Brunet et al., 1999). These results indicated that appropriate MI enhanced phosphorylated FoxO3 protein levels in muscle. Furthermore, as demonstrated in this study, the akt mRNA and protein levels support this hypothesis. Based on these findings, it can be concluded that the IGF-1/PI3K/AKT pathway regulates FoxO3 to negatively affect protein degradation, thereby inhibiting the expression of E3 ligase MAFbx and MuRF1.
Apart from protein deposition, lipids may play a significant role in the process of muscle hypertrophy. Lipids are signaling molecules and energy source for growth and reproduction, and the mass of lipids produced in muscle can regulate the muscle mass and energy metabolism of fish (Wang et al., 2023). In this study, appropriate MI levels increased lipid content in muscle tissue. Consequently, the increase in lipid content within muscle could potentially enhance energy metabolism in muscle, thereby facilitating muscle hypertrophy, and the specific mechanism necessitates further exploration.
In the above discussion, several possible mechanisms through which MI promotes skeletal muscle hypertrophy in fish were elaborated upon. Additionally, it has been discovered that there are also social network-like connections among these pathways. Firstly, the transcript factor binding sites are identified within the FST promoter region. Nrf2 can bind to the FST promoter region ARE, thereby activating FST transcription (Lin et al., 2016). Therefore, under the action of MI, FST may translocate to the nucleus and exert a certain role in the nucleus, thus influencing the number of myonuclear morphologies, which requires further verification. The second crosstalk exists between MSTN and the AKT/TOR pathway. Suryawan found that injection of FST into rat muscle inhibited MSTN and enhanced protein synthesis (Suryawan et al., 2006). In addition, mice knocked out of MSTN and mice treated with anti-myostatin antibodies also had increased protein synthesis (Welle et al., 2006). Based on the above findings, MI may affect the interaction between MSTN and AKT pathways and suggest that MSTN may be a key signaling molecule that governs protein deposition. The last crosstalk exists between IGF-1 and myogenic regulators. Research has demonstrated that IGF-1 can regulate the expression of MRFs in rainbow trout myoblasts, such as MyoD and MyoG (Garikipati and Rodgers, 2012). In summary, the effect of dietary MI on the growth and development of fish and fish myofibers are carried out within a network of extensive signaling pathways.
This study demonstrated that dietary MI supplementation improves adult grass carp growth performance and myofiber development (Fig. 6). The regulation of muscle development by MI is a complex process, and the present study unveiled several interesting findings. First, an increase in the number of myonuclear may be attributed to an important role of MI in the activation of the LOX/fibronectin/integrin signaling pathway. Second, the MSTN signaling pathway was inhibited by MI, possibly related to MI effect in promoting the elevated levels of FST protein. The inhibition of MSTN further regulates the expression of MRFs and facilitates the proliferation and differentiation of myoblasts, consequently influencing the growth and development of myofibers. Third, MI increased protein deposition in muscle. The synthesis of protein in muscle exceeds the degradation of protein, which promotes the growth and hypertrophy of fish muscle. According to the quadratic regression analysis of PWG, adult grass carp require 301.30 mg/kg MI in their diets.Fig. 6Schematic diagram of dietary myo-inositol promoting adult grass carp skeletal muscle hypertrophy. Na^+^/K^+^-ATPase = Na^+^/K^+^-adenosineadenosine-triphosphate; SMIT = sodium-myo-inositol cotransporter; IGF-1 = insulin-like growth factor-1; PI3K = phosphatidylinositol 3-kinase; AKT = protein kinase B; TOR = target of rapamycin; S6K1 = ribosomal protein S6 kinase 1; FoxO3 = forkhead box O3; MuRF1 = Muscle RING-finger protein-1; MAFbx = Muscle Atrophy F-box; MSTN = myostatin; MRF = myogenic regulatory factors; MyoD = myogenic determining; MyoG = myogenin; LOX = lysyl oxidase; Nrf2 = NF-E2-related factor 2.Fig. 6
Meiqi Wang: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Lin Feng: Visualization, Validation, Software, Resources, Methodology, Funding acquisition. Pei Wu: Project administration, Data curation, Conceptualization. Yang Liu: Methodology. Hongmei Ren: Methodology. Xiaowan Jin: Methodology. Xiaoqiu Zhou: Writing – review & editing, Supervision, Project administration, Funding acquisition. Weidan Jiang: Writing – review & editing, Supervision, Resources, Methodology, Investigation, Funding acquisition.
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.