Dihydromyricetin resists inflammation‐induced muscle atrophy via ryanodine receptor‐CaMKK‐AMPK signal pathway

Abstract Skeletal muscle plays a pivotal role in the maintenance of physical and metabolic health. Skeletal muscle atrophy usually results in physical disability, inferior quality of life and higher health care costs. The higher incidence of muscle atrophy in obese and ageing groups is due to increased levels of inflammatory factors during obesity and ageing. Dihydromyricetin, as a bioactive polyphenol, has been used for anti‐inflammatory, anti‐tumour and improving insulin sensitivity. However, there are no published reports demonstrated the dihydromyricetin effect on inflammation‐induced skeletal muscle atrophy. In this study, we first confirmed the role of dihydromyricetin in inflammation‐induced skeletal muscle atrophy in vivo and in vitro. Then, we demonstrated that dihydromyricetin resisted inflammation‐induced skeletal muscle atrophy by activating Ca2+‐CaMKK‐AMPK through signal pathway blockers, Ca2+ probes and immunofluorescence. Finally, we clarified that dihydromyricetin activated Ca2+‐CaMKK‐AMPK signalling pathway through interaction with the ryanodine receptor, its target protein, by drug affinity responsive target stability (DARTS). Our results not only demonstrated that dihydromyricetin resisted inflammation‐induced muscle atrophy via the ryanodine receptor‐CaMKK‐AMPK signal pathway but also discovered that the target protein of dihydromyricetin is the ryanodine receptor. Our results provided experimental data for the development of dihydromyricetin as a functional food and new therapeutic strategies for treating or preventing skeletal muscle atrophy.


| INTRODUC TI ON
Skeletal muscle comprises about 45% of the human body mass.
Obesity and ageing are often accompanied by skeletal muscle atrophy. 1 The main feature of skeletal muscle atrophy is a reduction in muscle mass and myofibre diameter. Skeletal muscle atrophy usually results in physical disability, inferior quality of life, and higher healthcare costs. 2 Since skeletal muscle plays an essential role in maintaining body flexibility, it is critical for us to understand the mechanism of skeletal muscle atrophy for maintaining optimal health throughout life.
The skeletal muscle mass depends on the balance between protein synthesis and degradation in myofibre. 3 The main signalling pathways that regulate skeletal muscle growth include the insulin signalling pathway responsible for protein synthesis and the ubiquitinproteasome system responsible for protein degradation. Insulin activates the IRS-1/PI3K/AKT pathway by binding to the insulin receptor. AKT activates the mammalian target of rapamycin (mTOR)/ S6 protein kinase (S6K) pathway increasing protein synthesis. 4 E3 ubiquitin protein ligase is a key enzyme of the ubiquitin-proteasome system,it recognizes the substrate and labels it by ubiquitination.
The proteasome will then degrade the labelled protein. Muscle ring finger protein 1 (MuRF1) and muscle atrophy F-box (atrogin-1) are two muscle-specific E3 ligases. MuRF1 and atrogin-1 induce hydrolysis of skeletal muscle structure proteins and myogenic differentiation transcription factors, leading to skeletal muscle atrophy. 5 The increasing incidence of skeletal muscle atrophy is closely related to the sharp increase in the obese population over the world.
Obesity is characterized by increased production of tumour necrosis factor-alpha (TNFα) and other pro-inflammatory factors. 6 Previous studies have shown that TNFα activated the c-Jun N-terminal kinase (JNK) pathway, induced the insulin resistance in skeletal muscle. Insulin resistance reduced protein synthesis in skeletal muscle. 7 Other studies have reported that TNFα activated inhibitor of nuclear factor-kappa B (IκB)/nuclear factor-kappa B (NF-κB) directly induced the expression of MuRF1 and atrogin-1. 8 In insulin-resistant individuals, the effect of AKT inhibition on forkhead proteins disappeared and ultimately increases the expression of MuRF1 and atrogin-1. 9 Since there is a close relationship between obesity-induced inflammation and skeletal muscle atrophy, preventing the skeletal muscle inflammation response in obese individuals will be a new strategy for reducing the incidence of skeletal muscle atrophy.
Dihydromyricetin (DHM), also known as ampelopsis, is the main bioactive polyphenol in rattan tea. Rattan tea has been used for antiinflammatory in China and other Asian countries for several centuries. 10 DHM exerted its anti-inflammatory effect in rats through suppressing NF-kB signalling in macrophage. 11 DHM also improved physical performance at high altitude by maintaining mitochondrial biogenesis in skeletal muscle. 12 DHM prevented skeletal muscle insulin resistance by inducing autophagy through the AMPK-PGC-1α-Sirt3 signalling pathway. 13 Currently, most DHM-related research reported that DHM functions depend on its activation of AMPK, but AMPK is not the direct target of DHM in cells, and the target of DHM in cells is still unclear. To our knowledge, there is no published paper demonstrated the DHM effect on inflammation-induced skeletal muscle atrophy.
We used a high fat diet (HFD)-induced obese mice model to study inflammation-induced skeletal muscle atrophy in vivo. Gavage DHM was used to determine DHM function in reducing the level of inflammation and inhibiting skeletal muscle atrophy in obese mice. C2C12 cells were treated with TNFα as a model of inflammation-induced muscle atrophy in vitro. We have elucidated the molecular mechanism of DHM for preventing inflammation-induced skeletal muscle atrophy in C2C12 cells using immunofluorescence, signal pathway blocking and drug affinity responsive target stability (DARTS). Our results provided not only experimental data for the development of DHM as a functional food but also provided new therapeutic strategies for the skeletal muscle atrophy.

| Animal experiment
Thirty-six 18-day-old specific-pathogen-free (SPF) healthy male C57B/L6 mice were purchased from the Animal Experiment Center of Guangdong Province. The mice were housed under a 12-h light and 12-h dark cycle (7 am and 7 pm, 25℃ and 70% ~ 80% humidity).
The mice were divided into two groups randomly: chow diet group (control, n = 12) and the HFD group (HFD, n = 24). Mice body weight gain was measured every Monday morning. After 8 weeks, the body composition of the mice, the strength of the skeletal muscles and the endurance exercise capacity of the mice were measured, to make sure the model of obesity-induced skeletal muscle atrophy was successfully constructed. Then, the obese mice were divided into two groups randomly: the HFD + PBS gavage (HFD) and the HFD + DHM gavage (DHM, purity by HPLC ≥ 98%, Shanghai Standard Biotech Co., Ltd.). Two hundred microliters of DHM (200 mg/kg body weight) was administered orally by gavage to the DHM group daily, while the control group and the HFD group were administered the same volume of PBS each day. We chose the rational dose of DHM (200 mg/kg body weight) according to the literature. 14,15 The body weight (BW) and feed intake of the mice were analysed every week.
At week 18, we analysed the body composition of mice and monitored the respiratory exchange rate and exercise frequency of mice in a metabolic cage. At week 19, we tested the mice's skeletal muscle grip strength and exercise endurance. At week 20, the mice were sacrificed to collect serum, gastrocnemius, tibialis anterior muscle and other tissues for further analysis.

| Haematoxylin-eosin staining (H&E)
The mice skeletal muscle was fixed in 4% formaldehyde (DaMao) at room temperature for 48 h. The method used for the H&E staining has been described previously. 16 The muscle fibre diameter was measured using Image-Pro Plus (IPP) 6.0 software (Media Cybernetics, Inc.).

| Oxygen consumption and exercise frequency assay
After mice were administered oral gavage with DHM for 10 weeks, O 2 consumption (VO 2 ), respiratory exchange ratio (RER), and exercise frequency of the mice were obtained by the promotion metabolism measurement system (Sable Systems International, USA).

| Strength and exercise endurance assay
The maximum muscle force measured 5 times by a grip strength meter (BIO-GS3, Bioseb/France), and the mean maximum strength of the twelve mice was used for data analysis of muscle strength.
The mice exercise endurance measured on the FT-200 Animal treadmill (Techman) at a speed of 10 m/min. Keep the mice running to exhaustion and record the time.

| Body composition analysis
Fat mass, lean mass, and body composition were determined using a nuclear magnetic resonance system according to the manufacturer's instruction (Body Composition Analyzer MiniQMR23-060H-I, Niumag Corporation).

| C2C12 cell culture and inflammatory induce C2C12 cell muscle atrophy
The C2C12 cell line used in this study was purchased from American Type Culture Collection (ATCC). C2C12 cells were cultured in DMEM/ HIGH GLUCOSE (Hyclone) with 10% foetal bovine serum (Gibco).
C2C12 cells were seeded in 24-well plates (4 × 10 5 /cm 2 ). After 24 h, we treated the C2C12 cells with TNFα (MedChemExpress, Monmouth Junction, USA) at the concentration of 1 ng/ml for 7 days to induce C2C12 cell muscle atrophy. Dissolve 3.2 mg DHM into 10 ml DMSO to prepare a 1 mM DHM solution. In C2C12 cell experiments, the DHM solution was mixed into the cell culture medium at a ratio of 1/1,000. We treated C2C12 cells with TNFα and DHM (1 μM, purity ≥ 98%, Sigma Chemical Inc.) for 7 days to demonstrate DHM resisted inflammation-induced muscle atrophy. We treated C2C12 cells with TNFα, DHM and Compound C (5 μM, MedChemExpress) for 7 days to demonstrate DHM resisted inflammation-induced muscle atrophy through AMPK. We treated C2C12 cells with TNFα, DHM and STO-609 (10 ng/ml, MedChemExpress) for 7 days to demonstrate DHM-resisted inflammation-induced muscle atrophy through CaMKK. We treated C2C12 cells with TNFα, DHM and ryanodine (100 nM, MedChemExpress) for 7 days to demonstrate DHM resisted inflammation-induced muscle atrophy through the ryanodine receptor.

| Glucose uptake assay
Seven days after treatments, the glucose uptake was assayed by 2-NBDG (MedChemExpress) according to the manufacturer's protocol. 2-NBDG is a fluorescent glucose analog that has been used to monitor glucose uptake in live cells. Therefore, the intensity of 2-NBDG immunofluorescence reflects glucose uptake and insulin sensitivity. C2C12 cells were incubated with or without media con-

| RNA Extraction and PCR Analysis
Methods used for the RNA extraction and PCR analysis have been described previously. 17 The relative expression of mRNAs was normalized with β-actin levels using the 2 −ΔΔCt method. 2 −ΔΔCt is defined as the ratio of the relative mRNA or miRNA level between the experimental group and the control group. Primers were designed using Primer Premier 5 based on sequences of mice genes obtained from NCBI. All the primers used in this study are shown in Table 1.

| Western blot analysis
The method used for the Western blot analysis has been described previously. 18 Band intensities were quantified by ImageJ software.
The antibodies and their dilutions used in this study are listed in Table 2.

| Immunofluorescent staining and confocal microscopy
C2C12 cell immunofluorescence staining was conducted after 7 days of treatments. The method used for MyHC and atrogin-1 immunofluorescent assay has been described previously. 19 The fluorescence was observed using Nikon Eclipse Ti-s microscopy (Nikon).
The cell nuclei were stained for DAPI (Beyotime).

| Drug affinity responsive target stability (DARTS) assay
Drug affinity responsive target stability experiments for identifying the targets of DHM were performed as previously reported. 20 In brief, C2C12 cells were lysed and treated with DHM (10 nM or 1 μM) for 1 h at room temperature. Then, the mixture was digested with 0.01% protease for 30 min at room temperature. The digestion was stopped by directly add 5×SDS-PAGE loading buffer and inactivation by boiling 5 min. Protein samples were separated with 8%-15% SDS-polyacrylamide gels and analysed by Coomassie blue staining and Western blotting.

| Statistical analysis
All data are expressed as the mean ± standard deviation (SD) of three independent experiments. Our data are a normal distribution, and the homogeneity of data between each group is equal under the SPSS analysis. In Figure 1A-D, I, K, L , Figure 3F and

| DHM reduced fat accumulation and inflammation in HFD-induced obese mice
In the HFD-induced obese stage, HFD significantly increased body weight gain in mice after eight weeks, compared with chow diet-fed mice ( Figure 1A). HFD also significantly increased body fat content in mice ( Figure 1B), indicating that we have constructed HFDinduced obese mice successfully. In addition, the muscle content was decreased in the HFD-induced obese mice compared with chow diet-fed mice ( Figure 1B). Both muscle strength ( Figure 1C) and endurance exercise capacity ( Figure 1D) were lower in HFD-induced obese mice compared with chow diet-fed mice. These results indicated that we have successfully constructed an obesity-induced skeletal muscle atrophy model.

Gene name
Forward primer sequence (5′−3′) Reversed primer sequence (5′−3′) In the DHM treatment stage, HFD increased energy intake and body weight gain in mice compared with the chow diet group, and DHM did not affect energy intake and body weight gain in our mice feeding experiments compared with the HFD group ( Figure 1E, F).
We also analysed the body composition of mice and found that HFD increased fat mass and decreased muscle mass compared with chow diet group ( Figure 1G). Compared with the HFD group, DHM prevented the reduction of muscle mass caused by HFD ( Figure 1G).
Subsequently, we monitored the respiratory exchange rate and exercise frequency of mice in a metabolic cage. We found that DHM feeding did not change exercise frequency ( Figure 1L

| DHM resisted inflammatory-induced skeletal muscle atrophy in mice
To investigate whether DHM resisted inflammatory-induced skeletal  muscle weight gain compared with the HFD group ( Figure 2D). The muscle fibre diameter of the gastrocnemius and tibialis anterior muscles were also reduced in the HFD-induced obese mice based on the H&E results. DHM reversed the inhibitory effect of HFD on muscle fibre growth ( Figure 2E-G). In addition, qPCR and Western blot results showed that AMPK, a skeletal muscle energy metabolism sensor, was inhibited in HFD-induced obese mice compared with the chow diet group ( Figure 2H, I and Figure S1). HFD-induced obesity activated the expression of NF-κB, the inflammatory responserelated gene; NF-κB, then, in turn, induced the expression of atrogin-1, the muscle atrophy-related protein ( Figure 2H, I and Figure S1).

| TNFα induced inflammatory response and muscle atrophy in C2C12 cells
We constructed a cellular model of TNFα-induced muscle atrophy in C2C12 cells to establish the mechanism of DHM in relieving HFDinduced inflammation and muscle atrophy. TNFα, less than 10 ng/ ml, did not damage cell viability using MTT assay compared with the control group ( Figure 3A). To simulate the low-level inflammatory response during obesity, we selected the TNFα concentration of 1 ng/ml for 5 days in the subsequent experiments. After 5 days of TNFα treatment, insulin stimulation did not increase glucose uptake in C2C12 cells, and the cells developed an insulin resistance phenotype ( Figure 3B and Figure S2A). Then, we measured the expression of astrogin-1, a muscle atrophy marker and myosin, a skeletal muscle differentiation marker by immunofluorescence. TNFα treatment increased (p < 0.05) the expression of astrogin-1 ( Figure 3C and Figure S2B) and inhibited (p < 0.05) the expression of myosin compared with control group ( Figure 3D and Figure S2C). Compared with the control group, the phosphorylation of AMPK was reduced, and the phosphorylation of NF-κB was increased after TNFα treatment, according to the Western blot results ( Figure 3E and Figure S2D).
The phosphorylation of NF-κB induced the expression of atrogin-1, an atrophy-related gene. TNFα treatment also decreased IRS-1 expression compared with the control group, an insulin sensitivityrelated gene ( Figure 3E, F and Figure S2D). The insulin resistance inhibited the phosphorylation of mTOR and inhibited myosin expression ( Figure 3E, F and Figure S2D). Thus, the above results indicated that TNFα activated the inflammatory response and induced muscle atrophy. At the same time, the inflammatory response caused insulin resistance to inhibit protein synthesis and eventually exacerbated muscle atrophy.

| DHM blocked expression of the inflammatory response-induced muscle atrophy-related genes
Then, we investigated whether DHM prevented inflammatory response-induced muscle atrophy. Compared with the control group, DHM, less than 3 μM, did not damage cell viability through the MTT assay ( Figure 4A). DHM treatment inhibited TNFα-induced atrogin-1 expression ( Figure 4B and Figure S3A), and DHM reversed the inhibitory effect of TNFα on myogenic development compared with TNFα group, as shown by the immunofluorescence results ( Figure 4C and Figure S3B). Compared with TNFα treatment, cotreatment with TNFα and DHM, AMPK was activated, and NF-κB was inhibited, as shown by the qPCR and WB results ( Figure 4D, E and Figure S3C). Compared with TNFα treatment, DHM inhibited the expression of atrogin-1 and MuRF1, two atrophy-related genes.
The inhibition of atrogin-1 and MuRF1 inhibited the degradation of myosin ( Figure 4D, E and Figure S3C). Based on these results, we conclude that DHM exerted its anti-muscle atrophy function by blocking the expression of inflammation-induced muscle atrophyrelated genes.

| DHM improved TNFα-induced insulin resistance and promoted protein synthesis in C2C12 cells
Improving myoblast insulin sensitivity and increasing myoblast protein synthesis also inhibited muscle atrophy. We then investigated  Figure S4B). Based on these results, we conclude that DHM improved insulin resistance caused by TNFα, promoted protein synthesis in C2C12 cells and finally exerted DHM function of resisting inflammation-induced muscle atrophy.

| DHM resisted inflammation-induced muscle atrophy through AMPK
We blocked AMPK activity using Compound C to investigate whether DHM exerted its anti-muscle atrophy function through AMPK in C2C12 cells. Compound C (5 μM) and DHM (3 μM) did not damage cell viability using MTT assay compared with the TNFα group ( Figure S5D). The ability of DHM to improve TNFα-induced insulin resistance was blocked by inhibiting AMPK, as shown by the glucose uptake assay ( Figure 6A and Figure S5A). The ability of DHM to prevent TNFα-induced muscle atrophy disappeared after blocking AMPK, as demonstrated by the immunofluorescence results ( Figure 6B and Figure S5B). When Compound C blocked AMPK activity, the activation effect of DHM on AMPK and mTOR disappeared ( Figure 6C and Figure

| DHM resisted inflammation-induced muscle atrophy through CaMKK-AMPK instead of LKB1-AMPK pathway
We blocked the AMPK upstream factors CaMKK and LKB1 to investigate the pathway DHM used to activate AMPK. When STO-609 blocked CaMKK activity, the ability of DHM to improve TNFαinduced insulin resistance disappeared, as shown by the glucose uptake assay ( Figure 7A and Figure S6A). The ability of DHM to prevent TNFα-induced muscle atrophy disappeared after CaMKK was blocked, as shown by the immunofluorescence results ( Figure 7B and Figure S6B). When STO-609 blocked CaMKK activity, the activation effect of DHM on CaMKK, AMPK and mTOR disappeared ( Figure 7C and Figure Figure S6D). LKB1 interference did not affect the activation of AMPK by DHM ( Figure 7F and Figure S6D). LKB1 interference did not affect the improvement of DHM on TNFα-induced insulin resistance, as shown by the glucose uptake assay ( Figure 7G and Figure S6E). LKB1 interference also did not affect the DHM ability in inhibiting TNFα-induced muscle atrophy, as demonstrated by the immunofluorescence results ( Figure 7H and Figure S6F). These results indicated that DHM resisted inflammation-induced muscle atrophy in C2C12 cells through CaMKK-AMPK instead of the LKB1-AMPK pathway.   Figure 8A and Figure S7A).

| DHM activated CaMKK by increasing intracellular Ca 2+ concentration through ryanodine receptor
After the cellular Ca 2+ was cleared by the intracellular Ca 2+chelating agent BATAP-AM, the activation effect of DHM on intracellular Ca 2+ was eliminated, indicating that DHM activation on intracellular Ca 2+ was dependent on intracellular Ca 2+ storage ( Figure 8B and Figure S7B). After cellular Ca 2+ was cleared, DHM could no longer activate CaMKK, as shown by the WB results ( Figure 8C and Figure S7C). Since endoplasmic reticulum was the major organelle for Ca 2+ release and recovery in myoblasts, we used ryanodine to block the endoplasmic reticulum Ca 2+ channel ryanodine receptor and U73122 to block the IP3 receptor.
Blocking the IP3 receptor pathway did not affect cellular activation of intracellular Ca 2+ signal induced by DHM ( Figure 8D and Figure S7D). After ryanodine blocked the ryanodine receptor, the DHM function on activating intracellular Ca 2+ signal disappeared ( Figure 8D and Figure S7D). After blocking the ryanodine  Figure 8E and Figure S7E). These results indicated that DHM activated CaMKK by increasing intracellular Ca 2+ concentration through the ryanodine receptor.

| DHM resisted inflammatory-induced muscle atrophy through interaction with ryanodine receptor
We verified the interaction of DHM and ryanodine receptor by drug affinity responsive target stability (DARTS) assay to investigate the pathway DHM used to activate the ryanodine receptor.  Figure S8A).  Figure S8A). When ryanodine blocked the ryanodine receptor, the ability of DHM to improve TNFα-induced insulin resistance disappeared, as shown by the glucose uptake assay ( Figure 9E and Figure S8B). The ability of DHM to prevent TNFα-induced muscle atrophy disappeared after the ryanodine receptor was blocked, as shown by the immunofluorescence results ( Figure 9F and Figure S8C). These results indicated that DHM resisted inflammatory-induced muscle atrophy through interaction with the ryanodine receptor.

| DISCUSS ION
Skeletal muscle plays a pivotal role in the maintenance of physical and metabolic health. 21 Skeletal muscle atrophy, characterized by muscle mass loss and function decline, is due to an increase of muscle protein degradation and reduction of protein synthesis. Muscle mass loss is frequently associated with inflammation, ageing, injury and obesity. 2 Therefore, therapeutic strategies for skeletal muscle atrophy need to block protein degradation and increase protein synthesis. In this study, we found that DHM activated the Ca 2+ -CaMKK-AMPK signal pathway by binding to the ryanodine receptor. Activation of AMPK increased protein synthesis by improving inflammation-induced skeletal muscle insulin resistance. AMPK also inhibited protein degradation by blocking the inflammation-induced skeletal muscle inflammatory response. Our study not only clarified the molecular mechanism of DHM resistance to inflammationinduced skeletal muscle atrophy but also discovered the target of DHM in this process. This study provided a new therapeutic strategy for the obesity-induced skeletal muscle atrophy.
The increasing incidence of skeletal muscle atrophy is inextricably related to the increase in the global obese population. 22 29 We demonstrated that DHM activated AMPK in vivo and in vitro, and we also showed that the function of DHM in blocking muscle atrophy depended on its activation effect on AMPK in vitro.
As the key regulator of cellular energy metabolism, AMPK inhibited anabolism to and promoted catabolism. 30 In this study, we AMPK not only regulated energy metabolism but also blocked the inflammatory response by inhibiting the NF-κB signalling pathway. After AMPK activation, SIRT1 was activated by the lowered NAD + level. P65, the subunit of NF-κB, was deacetylated by SIRT1; then, the transcriptional activity of NF-κB was also inhibited. 32 Inflammatory cytokines and fatty acids activated NF-κB signalling F I G U R E 7 DHM (3 μM) resisted inflammation-induced muscle atrophy through CaMKK-AMPK instead of the LKB1-AMPK pathway. (A) DHM alleviated TNFα-induced insulin resistance was dependent on CaMKK, as shown by the glucose uptake test. (B) DHM alleviated TNFα-induced muscle atrophy was dependent on CaMKK, as shown by the immunofluorescence results. Blue represents the nucleus, and red represents atrogin-1. (C) DHM regulated the protein level of inflammatory response-induced muscle atrophy-related genes was dependent on CaMKK, as shown by WB. "p-" before the gene name means phosphorylated form. (D) DHM regulated the mRNA level of inflammatory response-induced muscle atrophy-related genes was dependent on CaMKK, as shown by qPCR. (E) LKB1 was interfered with by small RNA, as shown by qPCR. (F) LKB1 interference did not affect the AMPK expression, as shown by WB. "p-" before the gene name means phosphorylated form. (F) LKB1 did not affect DHM improvement on TNFα-induced insulin resistance, as shown by the glucose uptake test.
(G) LKB1 did not affect DHM improvement on TNFα-induced muscle atrophy, as shown by the immunofluorescence results. Blue represents the nucleus, and red represents atrogin-1. N = 6, ** p < 0.01. Bars with different letters indicate they are significantly different (p < 0.05) Dandelion root extract (10-400 µg/ml) dose dependently increased intracellular Ca 2+ level in the presence of external Ca 2+ . The dandelion root extract-induced Ca 2+ increase was significantly reduced in the absence of extracellular Ca 2+ . 40 However, in our study, DHM activated intracellular Ca 2+ signals regardless of the presence or absence of extracellular Ca 2+ . The endoplasmic reticulum is the main storage organelle for calcium ion in skeletal muscle. The ryanodine receptor and IP3 receptor are the main channels for calcium release from the endoplasmic reticulum and play a central role in excitation-contraction coupling in skeletal muscle. 41 In this study, we discovered that blocking the ryanodine receptor, the DHM's ability to activate Ca 2+ and CaMKK disappeared, but blocking the IP3 receptor had no effect on preventing the activation of Ca 2+ -CaMKK induced by DHM.

F I G U R E 8 DHM (3 μM) activated CaMKK by increasing intracellular Ca
The identification of target protein for small molecules is critical for chemical metabolomics and drug discovery. 42 DARTS is a straightforward and quick approach to identify potential target protein of small molecules. The mechanism of DARTS is that the interaction of target protein and small molecules resists proteolysis. The most significant advantage of this method is it allows the use of the small native molecule without any immobilization or modification, such as the incorporation of biotin, radioisotope or photo-affinity labels. 20,43 In this study, we used DARTS to identify potential binding targets of DHM and used DARTS-Western blotting to test and validate the potential DHM target. Our results showed that DHM interacted with ryanodine receptor protein to activate the Ca 2+ -CaMKK-AMPK signal pathway.
In conclusion, our results not only show that DHM resisted inflammation-induced muscle atrophy but also demonstrate that the DHM activated the Ca 2+ -CaMKK-AMPK signal pathway through interacting with its target protein ryanodine receptor ( Figure 10). Our F I G U R E 1 0 Summary model of dihydromyricetin resists inflammationinduced muscle atrophy via the ryanodine receptor-CaMKK-AMPK signal pathway results provided experimental data for the development of DHM as a functional food and new therapeutic strategies for treating or preventing skeletal muscle atrophy.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

CO N S E NT FO R PU B LI C ATI O N
All authors have read and approved for publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
We confirm that the data supporting the findings of this study are available within the article and its supplementary materials.