Deciphering the Molecular Mechanism of Yifei-Sanjie Pill in Cancer-Related Fatigue

Background The incidence of cancer-related fatigue (CRF) is increasing, but its lack of clear pathogenesis makes its prevention and treatment difficult. Therefore, it is of great significance to clarify the pathogenesis of CRF and find effective methods to treat it. Methods The CRF model was established by intraperitoneal injection of LLC cells in ICR mice to explore the pathogenesis of CRF and verify the therapeutic effect of the Yifei-Sanjie pill (YFSJ). The active components of YFSJ were found by LC/MS, the in vitro inflammatory infiltration model of skeletal muscle was constructed by TNF-α and C2C12 myoblasts, and the results of in vivo experiments were verified by this model. Results Behavioral analysis results showed that YFSJ alleviated CRF; histological examination results showed that YFSJ could reverse the tumor microenvironment leading to skeletal muscle injury; ELISA and RNA-seq results showed that the occurrence of CRF and the therapeutic effect of YFSJ were closely related to the tumor inflammatory microenvironment; IHC and WB results showed that the occurrence of CRF and the therapeutic effect of YFSJ were closely related to the Stat3-related signaling pathway and autophagy. Conclusions YFSJ can reduce the level of inflammation in the tumor microenvironment in vivo, inhibit the abnormal activation of the Stat3/HIF-1α/BNIP3 signaling pathway induced by tumor-related inflammation, thereby inhibiting the overactivation of mitophagy in skeletal muscle, and finally alleviate CRF. Quercetin, one of the components of YFSJ, plays an important role in inhibiting the phosphorylation activation of Stat3.


Introduction
With the development of society and the improvement of medical level, people's life expectancy is getting longer and longer [1]. Cancer, as a disease closely related to the aging of the body, is accompanied by an increase in life expectancy, and the incidence of cancer is increasingly high [2,3]. Although the lesions of cancer are mostly confned to one or more places, in the process of their occurrence and development, they have an obvious infuence on the whole body.
In the process of cancer development, there are a variety of unpleasant symptoms, such as fatigue, weight loss, and pain. Te incidence of cancer-related fatigue (CRF) has been reported to be as high as 82% in cancer patients, and tumor burden contributes to varying degrees of fatigue with or without chemotherapy [4]. Unfortunately, the pathogenesis of CRF is not fully understood, and there is no standard treatment.
Previous studies have found that the mechanism of CRF is closely related to tumor-induced overactivation of mitophagy in skeletal muscle [4]. Tere are many reasons for excessive mitophagy, such as activation of infammatory factors, oxidative stress, and energy metabolism disorders [5][6][7]. Tumor burden will also lead to changes in the body's internal environment, such as changes in the immune microenvironment and energy metabolism level [8,9]. However, the mechanism by which tumor burden leads to mitophagy and fatigue remains unclear.
Te Yifei-Sanjie pill (YFSJ), a traditional Chinese patent medicine, can be used to alleviate CRF in clinical practice [10][11][12]. Te mechanism may be to alleviate CRF by reducing skeletal muscle damage [4,13]. However, the specifc molecular mechanism by which YFSJ attenuates skeletal muscle injury in the tumor microenvironment has not been revealed. Terefore, it is of great signifcance to clarify the relationship between the occurrence of CRF and mitophagy and to reveal the molecular mechanism of YFSJ in the treatment of CRF. In addition, meta-analysis and network pharmacology predicted that quercetin may play an important role in alleviating CRF, but its specifc molecular mechanism has not been revealed [14]. Terefore, we will further study the pathogenesis and treatment of CRF.
In this study, we demonstrated that the tumor infammatory microenvironment leads to the abnormal activation of the Stat/HIF-1α/BNIP3 signaling pathway in skeletal muscle cells, which induces excessive mitophagy in skeletal muscle and ultimately leads to fatigue. Several miRNAs have previously been reported to regulate these pathways, but the mechanisms are unclear [15]. At the same time, we confrmed that YFSJ could reduce tumorinduced elevated levels of infammation in vivo, thereby inhibiting excessive mitophagy in skeletal muscle and ultimately alleviating CRF. Meanwhile, we also found that quercetin, a major component in YFSJ, can specifcally inhibit the phosphorylation activation of Stat3, thereby inhibiting TNF-α-induced mitophagy, which provides a new paradigm for the modern development and application of traditional drugs.

Animal Ethics Statement.
Female ICR mice weighing between 20 and 22 g and aged 4 weeks (Charles River Laboratories, Zhejiang, China) were used in this investigation. All experiments were conducted according to the relevant laws and institutional guidelines. Mice were individually housed in independent ventilated cages at 24°C to 26°C under constant humidity with a 12 h light/dark cycle. Based on clinical practice, the mice were divided into 4 groups (n � 10), namely, control, CRF, low, and high groups, in a random order. Permission for the experimental scheme from the Laboratory Animal Ethics Committee of Jinan University was granted (Approval No. 20220301-15).

YFSJ Preparation
. YFSJ (Cat. #Z20190015000) was purchased from the First Afliated Hospital of Guangzhou University of Chinese Medicine (Guangdong, China). Each packet of YFSJ was 8 g, which was equivalent to 16.4 g of herbal medicine. Te dosage of the subsequent experiments was the amount of herbal medicine.

In Vivo Infammatory CRF Model.
We induced an infammatory CRF model in mice via intraperitoneal injection of LLC cells following the published protocols [16]. After acclimation for 7 days, mice in groups CRF, Low, and High were intraperitoneally injected with 1 × 10 7 LLC cells/100 μL each mouse. Te experimental protocol is shown in Figure 1(a). Tree days after LLC cell inoculation, treatment was initiated, and the dose of YFSJ was referred to in our previous report [4]. Te control and CRF groups were given normal saline intragastric administration (0.2 ml/d) for 28 days. Low group was given 2 g/kg/d YFSJ by gavage, 0.2 ml, for 28 days. High group was given 4 g/kg/d YFSJ by gavage, 0.2 ml, for 28 days.   Journal of Oncology a force transducer, then let the mouse grasp the grid tightly with both forepaws, and then pulled the mouse away from the grid, breaking its grasp. Te transducer records the maximum force applied by the mouse on the grid during the pull. Let the mouse pull the grid three times in a row, and take the average of the three pulls.

Weight-Loaded Exhaustive Swimming Test (WEST).
Te muscle exercise endurance of mice was measured by WEST according to the published protocol [18]. In short, the mice were individually loaded with a lead (7% of body weight) on their tail root and placed in the swimming pool (diameter 30 cm × height 50 cm) flled with 30 cm deep water (maintained at 25 ± 1°C). Te exhaustive swimming time was recorded as described in the previous study [19]. Considering that the mice could not fully recover their physical strength before the test in a short time, the test was not repeated for each mouse in a single test.

Open Field Test (OFT).
Te locomotor willingness and ability of mice were measured by OFT according to the published protocol [20]. In short, the OFT was conducted in an arena made of plexiglass (100 × 100 × 50 cm 3 ). Each mouse was placed in the center of the apparatus and analyzed for 10 min in a quiet room. Te total movement distance and movement speed were calculated and analyzed using the EthoVision XT 14 software (Noldus Information Technology Co., Ltd., Beijing, China). In consideration of the adaptation of mice to the experimental environment, the test was not repeated for each mouse in a single test.

Mouse Euthanasia and Sample Collection.
According to the corresponding experimental plan, the mice were anesthetized with pentobarbital (150 mg/kg, i.p.) after all the corresponding behavioral tests were completed (four mice at 14 days and six at 28 days). In order to maintain a sufcient number of mice for behavioral analysis, only 4 mice from each group were sampled at 14 days. Te plasma was collected, the mice were euthanized by cervical dislocation, and the lung, liver, spleen, and gastrocnemius muscle tissues were removed for subsequent experiments. Te plasma and tissues collected will be pretreated diferently depending on the subsequent experiments.  (iii) the OFT movement distance; (iv) the OFT movement speed. Te data are presented as means ± SD; ns * p > 0.05, * * p < 0.01, and * * * p < 0.001 compared with the control group; ## p < 0.01 and ### p < 0.001 compared with the CRF group; ns& p > 0.05 and && p < 0.01 compared with the low group; 0, 7, and 14 days, n � 10; 21 and 28 day, n � 6.

Hematoxylin and Eosin (HE)
Staining. HE-stained sections of lung, liver, and kidney were prepared according to previously reported methods [21]. Te stained slices were observed and photographed under a light microscope at 100× or 40× magnifcation (NIKON Eclipse ci, Japan). For quantifcation of immunostaining intensities, Image J software (National Institutes of Health, Bethesda, MD, USA) was used, as stated by Sysel et al. [22]. Te inverse mean density was determined, as reported by Vis et al. [23], in 1 randomly chosen feld from various sections of 3 mice in each group.

Transmission Electron Microscope (TEM)
. TEM sections of gastrocnemius muscle were prepared according to previously reported methods [4]. Te slices were observed and photographed under an electron microscope (HITA-CHI HT7700, Japan).

RNA-Seq.
Te mouse gastrocnemius muscle RNA-seq assay was performed as previously reported [21]. Te diferential expression analysis and diferential gene enrichment analysis (Gene Ontology, GO, and Kyoto Encyclopedia of Genes and Genomes, KEGG), and visualized, were performed on the Novo Magic (URL: https://magic.novogene.com) of Beijing Novogene Biotechnology Co., LTD.; n � 3. Datasets for RNA-seq can be obtained from the Sequence Read Archive at the NCBI (URL: https://www.ncbi.nlm.nih.gov/ sra/PRJNA874361).

Western Blotting (WB)
. WB analysis of gastrocnemius muscles was performed as previously reported [21]. If mitochondrial protein isolation is required, it is done using the . After the primary antibody protocol was completed, the corresponding secondary antibody (1 : 5000) was added according to the protocol. Te density values of the bands were captured and documented through a gel image analysis system (ChemiDox ™ , Bio-Rad, USA) and normalized to GAPDH or COX IV; n � 3.

Q-Orbitrap High Resolution Liquid/Mass Spectrometry (LC/MS).
As previously reported [4], we identifed the components contained in YFSJ by LC/MS. Tis part was completed by Wuhan Servicebio Company (Hubei, China).

Access to Active Ingredients and Molecular Docking.
LC/MS results were used in conjunction with previously reported methods [24] to obtain the active components in YFSJ. Using the TCM Network Pharmacology Analysis System (TCMNPAS, URL: https://54.223.75.62:3838/), quercetin was used as ligands and Stat3 as receptors for molecular docking, and their binding sites and binding afnity were analyzed. Protein docking pocket parameters were obtained from ligands, and the lower the value of "afnity" (the greater the absolute value), the stronger the binding force. Finally, all the docking results were analyzed by "RMSD," and an RMSD value less than 2 was considered reliable.

MDC Staining Assay.
Cell autophagy was measured by the MDC staining assay according to the manufacturer's instructions. Autophagy staining assay kit with MDC (Cat. #C3018S) was supplied by Beyotime Biotechnology (Shanghai, China). Te fuorescence intensities were measured at an emission wavelength of 512 nm and an excitation wavelength of 335 nm using a fuorescence microplate reader (BioTek, Vermont, USA). Te data are expressed as the percentage of the fuorescence intensity relative to that of the control group, n � 6.

Statistical Analysis.
Te experimental data were analyzed using Student-Newman-Keuls (S-N-K) in ANOVA with SPSS version 13.0 software (SPSS Inc., IL, USA) and GraphPad Prism 9 (GraphPad Software, LLC, California, USA) to graph the data. Te results are presented as the mean values ± standard deviation (SD). A p value <0.05 was considered statistically signifcant. Figure 1(a). In order to explore the dynamic changes of biochemical indexes in vivo during tumor development, mice were sampled at two time points in this experiment. At the end of the experiment, there was no signifcant diference in body weight between the groups (Figure 1(b)). Te results of FGST, refecting exercise intensity; WEST, refecting exercise endurance; and OFT, refecting exercise willingness and ability, showed that all aspects of the exercise level indexes in the CRF group were signifcantly lower than those in the control group (Figures 1(c)-1(e)). At the beginning or after the beginning of the treatment, each fatigue index of mice gradually showed the fatigue state of mice. After the YFSJ treatment, all the indexes refecting exercise were signifcantly improved. However, there was no signifcant diference in the efect of low-dose YFSJ and high-dose YFSJ, and only the total locomotion distance of the high group was higher than that of the low group in the OFT. Tese behavioral exercise indicators are important indicators to evaluate fatigue in various aspects. Terefore, our data show that the CRF model is successfully constructed, and YFSJ can efectively alleviate CRF.

YFSJ Reversed Cancer-Induced Mitochondrial Damage in Skeletal Muscle and Infammation Levels.
In order to explore the causes of CRF and the therapeutic efects of YFSJ, we analyzed the samples taken from mice, and the photos of mice before sampling are shown in Figure 2(a). HE staining showed tumor lesions in the lungs and liver of mice after intraperitoneal injection of LLC cells. In addition, LLC cell injection resulted in spleen enlargement and structural disorders (such as germinal center structural changes). High-dose YFSJ treatment could reverse spleen enlargement, but YFSJ has no obvious efect on the reversal of spleen substructural changes (Figure 2(b)). TEM showed that the structure of skeletal muscle fbers was disordered, the sarcoplasmic reticulum was dilated, and the number of mitophagosomes increased signifcantly in the CRF group. In the CRF group, muscle fber arrangement was more disordered and structure was more loose at 28 days compared with 14 days. After YFSJ treatment, the number of mitophagosomes in skeletal muscle was signifcantly reduced, and the morphology of muscle fbers was signifcantly improved (Figure 2(c)). ELISA results showed that the serum levels of proinfammatory factors, such as IL-6 and TNF-α, were signifcantly increased in the CRF group, and the level of TNF-α in the CRF group was signifcantly higher at 28 days than at 14 days. YFSJ could signifcantly reverse the increase in proinfammatory factor level caused by the tumor, and the reverse efect was more obvious in the high group than in the low group (Figures 2(d) and 2(e)). Based on these results, we suggested that the occurrence of CRF and the therapeutic efect of YFSJ are highly correlated with the tumor infammatory microenvironment and skeletal muscle mitophagy.

Te Cause of CRF and the Mechanism by Which YFSJ
Alleviates CRF Are Closely Related to Infammation, Mitophagy, and Stat/HIF-1 Signaling Pathway. To explore the potential mechanism of YFSJ in alleviating CRF, RNAseq was performed to identify the diferentially expressed transcripts (DETs) in skeletal muscle tissues among diferent groups. As shown in Figures 3(a) (i) and 3(a) (ii) (left panels), the CRF group signifcantly upregulated the expression of 461 transcripts and downregulated the expression of 612 transcripts in skeletal muscle tissues of mice, compared to the control group, while YFSJ treatment signifcantly upregulated the expression of 776 transcripts and downregulated the expression of 1074 transcripts, compared with the CRF group (Figures 3(b) (i) and 3(b) (ii), right panel).
GO enrichment analysis based on the DETs between control, CRF, and high groups revealed multiple predicted potential functions. As shown in Figure 3(a) (iii), biological process (BP) analysis showed that DETs were mainly associated with intracellular signal transduction, programmed cell death, and cellular immunity. Cellular component (CC) analysis indicated that DETs were mainly involved in the structure of mitochondria. In the molecular function (MF) category, DETs were signifcantly enriched in signaling receptor binding, cytokine and hydrolase activity, and so on. As shown in Figure 3(b) (iii), BP analysis showed that DETs were associated with intracellular signal transduction, programmed cell death, proteolysis, and immune response. As to CC, DETs were signifcantly enriched in the mitochondrial membrane part. Te MF analysis for these DETs includes lyase and cytokine activity, antioxidant activity, and signal transducer activity.
KEGG pathway analysis based on the DETs between the control and CRF groups revealed multiple enriched signaling pathways, including phagosome, mitophagy, oxidative phosphorylation, TNF signaling pathway, JAK-STAT signaling pathway, and HIF-1 signaling pathway (Figure 3(a) (iv)). Moreover, KEGG pathway analysis based on the DETs between the CRF and YFSJ groups demonstrated multiple enriched signaling pathways, including lysosome, mitophagy, oxidative phosphorylation, TNF signaling pathway, cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, and HIF-1 signaling pathway (Figure 3(b) (iv)). Te integrative analysis between the two comparisons (control vs. CRF and CRF vs. high) demonstrated that multiple signaling pathways were enriched in both comparisons. Notably, we fnd that both cytokine-cytokine receptor interaction, oxidative phosphorylation, and phagosome were signifcantly enriched in both comparisons, which encouraged us to further explore the regulatory efects of YFSJ on infammatory cytokines, mitophagy, and activation of the Stat/HIF-1/mitochondria signaling pathway in skeletal muscle tissues of CRF mice.  Journal of Oncology regulation of programmed cell death regulation of hydrolase activity intracellular signal transduction cell surface receptor signaling pathway immune response transmembrane receptor protein tyrosine kinase signaling pathway immune system process proteolysis regulation of signal transduction regulation of intracellular signal transduction mitochondrion mitochondrial part mitochondrial envelope microtubule associated complex mitochondrial membrane respiratory chain mitochondrial respiratory chain mitochondrial inner membrane mitochondrial protein complex mitochondrial membrane part signaling receptor binding tumor necrosis factor receptor binding cytochrome-c oxidase activity signal transducer activity cytokine activity singnaling receptor activity transmembrane signaling receptor activity peptidase activity, acting on L-amino acid peptides hydrolase activity, acting on carbon-nitrogen (but not peptide)bonds hydrolase activity, acting on glycosyl bonds ( Figure 4(a)). To further confrm the increased level of autophagy in skeletal muscle mitochondria in the CRF group and the efective reversal of excessive mitophagy by YFSJ, total protein and mitochondrial protein were extracted from skeletal muscle tissues for analysis. WB analysis of total protein of skeletal muscle tissue indicated that P-Stat3 and HIF-1α protein expression levels were increased in the CRF group, and YFSJ treatment signifcantly reversed these changes, and the reversal efect in the high group was more obvious than that in the low group. Moreover, WB analysis of mitochondrial proteins in skeletal muscle tissue indicated that HIF-1α, BNIP3, Beclin 1, Atg7, and LC3B protein expression levels were increased, while p62 protein expression levels were decreased in the CRF group. Similarly, YFSJ treatment reversed these changes in the CRF group, and the efect was more pronounced in the high group than in the low group (except for p62) (Figures 4(b)-4(d)). Terefore, these results indicated that YFSJ can attenuate tumor-induced infammatory infltration and overactivation of mitophagy in skeletal muscle tissue.

Quercetin Is an Active Component of YFSJ Acting on the
Stat3-Related Signaling Pathway. Te LC/MS results of YFSJ are shown in Figure 5(a). By comparing the LC/MS results with the database, we obtained quercetin, one of the active components of YFSJ ( Figure 5(b)). We used TCMNPAS to perform molecular docking between quercetin and Stat3 protein and verifed the docking results, which were visualized using software PMV-1.5.7 (DeLano Scientifc LLC). Te 3D structures of quercetin and Stat3 protein and its phosphorylation sites are shown in Figures 5(c) and 5(d).
Molecular docking results show that the binding site of quercetin to Stat3 is highly coincident with the phosphorylation site of Stat3, and the "absolute value of afnity" is relatively large, indicating that the binding of the two is relatively stable. We conducted RMSD verifcation on the predicted molecular docking modes of quercetin and Stat3, and the results showed that the RMSD values of all the predicted docking modes are less than 2. Tis suggests that the binding of quercetin to the phosphorylation site of Stat3 is feasible at the level of molecular interaction. In summary, we predict that quercetin, an active component in YFSJ, can efectively and specifcally inhibit Stat3 phosphorylation, thereby inhibiting the overactivation of the Stat3/HIF-1α/ BNIP3 signaling pathway caused by abnormal phosphorylation of Stat3, and ultimately inhibiting excessive mitophagy.

Quercetin Can Reverse TNF-α-Induced Mitophagy in C2C12
Myoblasts. In order to determine which infammatory factors activate mitophagy and whether quercetin can inhibit mitophagic cell death in skeletal muscle induced by infammatory factors, we used C2C12 myoblasts to establish an in vitro skeletal muscle model. MTT colorimetric results showed that TNF-α inhibited the viability of C2C12 myoblasts much more than IL-6 (Figures 6(a) and 6(b)). Terefore, TNF-α was selected for subsequent in vitro experiments. Although both YFSJ and quercetin alone had efects on the viability of C2C12 myoblasts, their efects were minor (Figures 6(c) and 6(d)). Our results showed that YFSJ, quercetin, and 3-MA could reverse the inhibitory efect of TNF-α on C2C12 myoblasts to a large extent (Figure 6(e)). 3-MA was used here as a positive control for the inhibition of autophagy. MDC autophagy staining showed that TNF-α could signifcantly induce autophagy in C2C12 myoblasts, which could be reversed by YFSJ, quercetin, and 3-MA ( Figure 6(f )). Te results of MDC autophagy staining would be even more signifcant if considering the decrease in cell number caused by TNF-α inhibition of C2C12 myoblast proliferation.
To further confrm that TNF-α can induce mitophagy in C2C12 myoblasts, thereby inhibiting the proliferation of C2C12 myoblasts. We used WB to analyze the total protein and mitochondrial protein of C2C12 myoblasts treated with each factor. WB results showed that the expressions of P-Stat3 and HIF-1α in total proteins increased signifcantly after TNF-α treatment, which was reversed by YFSJ and quercetin treatment, but not by 3-MA treatment. Moreover, TNF-α treatment increased the protein expression of HIF-1α, BNIP3, Beclin1, Atg7, and LC3B and decreased the protein content of p62 in mitochondria. YFSJ and quercetin treatment signifcantly reversed these changes, and 3-MA also reversed these changes (except HIF-1α and BNIP3) (Figures 6(g)-6(i)). Tese evidence suggest that TNF-α can efectively induce mitophagy in C2C12 myoblasts, thereby inhibiting the proliferation of C2C12 myoblasts, while YFSJ and quercetin can restore the viability of C2C12 myoblasts by reversing TNF-α induced mitophagy.

Discussion
CRF is an uncomfortable symptom associated with the occurrence and development of cancer, which seriously afects the quality of life of cancer patients [25]. Because the mechanism of CRF is not completely clear, it brings difculties in the clinical prevention and treatment of the disease. So far, there is no accepted treatment method for CRF. Ethnomedicine, represented by traditional Chinese medicine (TCM), is considered a potential treatment for CRF because TCM has many prescriptions for fatigue [26,27]. According to our study, we demonstrated that Chinese medicine YFSJ can efectively alleviate CRF. Meanwhile, the pathogenesis of CRF, the efect target, and the underlying molecular mechanism of YFSJ were revealed.
In the process of tumor growth, the body will produce a lot of proinfammatory factors [28], making the body of cancer patients always in a state of high infammation level [29]. Tis result was consistent with our studies. Our in vivo results demonstrated that the growth of tumor cells leads to an increase in the level of proinfammatory cytokines in serum, which in turn increases the level of infammatory infltration of muscle cells. YFSJ treatment can efectively reduce the level of proinfammatory factors in serum, thereby reducing the degree of infammatory infltration of muscle cells. At the same time, the size of the spleen can also refect the level of infammation in vivo to a certain extent [30]. We also found that the spleen of mice was signifcantly       enlarged after tumor cell inoculation and decreased after high-dose YFSJ treatment compared with the CRF group. Mitochondria are important places for energy production in the body. Te low mitochondrial function will lead to a lack of enough energy in the body, resulting in decreased exercise ability and a sense of fatigue [31]. We observed that the number of mitophagosomes was positively correlated with the degree of fatigue in mice. Our data confrmed that after the injection of tumor cells, all aspects of the motor indexes of mice showed varying degrees of decline, which directly indicated that the growth of tumor cells would cause fatigue in the host body. Fortunately, after YFSJ treatment, all exercise indexes refecting fatigue degree were improved to a great extent, which indicated that YFSJ could efectively alleviate CRF.
High levels of infammation in the body lead to mitophagy in skeletal muscle [32]. Our previous studies also confrmed that the occurrence of CRF is closely related to the damage of skeletal muscle [4]. Studies have shown that the tumor infammatory microenvironment can continuously activate STAT3-related signaling pathways [33]. Terefore, we linked infammation, Stat3 molecular-related signaling pathways, and mitophagy with the occurrence of CRF and the therapeutic efect of YFSJ. Activation by Stat3 phosphorylation promotes HIF-1α expression in the cytoplasm [34], and the HIF-1α in the cytoplasm binds to the mitochondrial membrane [35], further promoting the expression of BNIP3 in the mitochondria [36]. Te BH3 domain of BNIP3 competes with Beclin 1 to bind Bcl-2 or Bcl-xL, resulting in the formation of complexes between Beclin 1 and Class III PI3K, which activates mitophagy. At the mature stage of the autophagosome, BNIP3 can recruit the autophagy-related protein LC3 to the mitochondria. It promotes the formation of autophagosomes and actively participates in the clearance of damaged mitochondria [37]. Atg7 is a key component involved in autophagosome formation [38], and p62 is a marker of autophagic lysosomes. When autophagy occurs, lysosomes are degraded, and the expression of p62 will decrease [39]. Our data confrm that the tumor infammatory microenvironment in vivo abnormally activates the Stat3/HIF-1α/BNIP3 signaling pathway in skeletal muscle, leading to excessive mitophagy, which disrupts the body's energy balance and ultimately leads to fatigue.
To further validate the in vivo results and simplify the experimental model, we treated C2C12 myoblasts with TNFα to mimic the infammatory infltration of skeletal muscle in vitro. Consistent with our in vivo results, TNF-α signifcantly induced mitophagy and inhibited cell viability in C2C12 myoblasts. YFSJ and quercetin, the active component of YFSJ, signifcantly reversed TNF-α-induced mitophagy and cell viability inhibition in C2C12 myoblasts. 3-MA is an inhibitor of PI3K. It is widely used as an inhibitor of autophagy by inhibiting class III PI3K [40]. Terefore, it is able to prevent BNIP3 from further activating Beclin1, ultimately inhibiting the occurrence of autophagy. Data showed that 3-MA reversed TNF-α-induced autophagy in C2C12 myoblasts. Tese evidence further confrmed that TNF-α plays an important role in activating mitophagy, leading to CRF, and quercetin plays an important role in YFSJ treatment of CRF.
In summary, YFSJ acts as a traditional Chinese medicine compound, which may alleviate CRF through the action of multiple targets (Figure 7). Firstly, it reduces the level of proinfammatory factors in the body, thereby reducing the degree of infammatory infltration of skeletal muscle cells. Secondly, it inhibits the overphosphorylation of Stat3 protein (quercetin, an active ingredient in YFSJ, may be at work here), thereby inhibiting the overactivation of the Stat3/HIF-1α/BNIP3 signaling pathway. Finally, it inhibits excessive mitophagy and alleviates CRF under the action of multiple targets. Our results not only confrm that YFSJ is an efective  5 μM), and 3-MA (pretreatment with 2 mM for 4 h) on TNF-α (20 ng/ml)-induced decline viability and (f ) increase autophagy in C2C12 myoblast, n � 3. (g) WB analysis was performed to determine the protein expression of the Stat3/HIF-1α/BNIP3 signaling pathway and mitophagy related proteins, and (h, i) their relative expression levels. GAPDH or COX IV was used as the internal control. Data were presented as mean ± SD; ns * p > 0.05, * * p < 0.01, and * * * p < 0.001 compared with the blank group; ns# p > 0.05, ## p < 0.01, and ### p < 0.001 compared with the TNF-α group; n � 3.
regimen for the treatment of CRF but also reveal the pathogenesis of the disease and, at the same time, provide new clues for the development of drugs with natural compounds from ethnomedicine.

Conclusions
YFSJ and its component quercetin can inhibit the overactivation of skeletal muscle mitophagy mediated by the abnormal activation of the Stat3/HIF-1α/BNIP3 signaling pathway induced by the tumor infammatory microenvironment, thereby alleviating CRF.