Screening Of Active Ingredients Of Traditional Chinese Medicine Based On Stat3 Inhibition And The Therapeutic Effect On Cancer Cachexia

Cancer cachexia is a multifactorial disease syndrome characterized by weight loss and skeletal muscle atrophy.Several studies have also shown that preserving muscle mass can prolong the survival of patients and mice with cancer cachexia.However,there are currently no effective medical interventions to completely reverse cachexia,and there are no approved drug therapies.The continuous weight loss of cancer cachexia is mainly caused by skeletal muscle atrophy,and the mechanism of skeletal muscle atrophy is not fully clarified.At present,it is generally believed that skeletal muscle atrophy is mainly due to the interaction between tumor and the immune system,releasing excessive inflammatory cytokines such as interleukin 6(IL-6),tumor necrosis factor-alpha(TNF-?)and interleukin 1?(IL-1?),activate multiple signal transduction pathways in skeletal muscle,including signal transducer and activator of transcription 3(STAT3),nuclear factor-kappa B(NF-?B),and forkhead box protein O3(Fox O3))that ultimately activates muscle protein degradationubiquitin-proteasome system(UPS),promotes protein catabolism and muscle wasting.STAT3 is a transcription factor,which has many biological functions such as regulating cell proliferation,migration,survival and apoptosis.Recent studies have shown that in various animal models of cancer cachexia include chronic renal failure and diabetes,skeletal muscle atrophy is closely related to STAT3 activation;further mechanism studies found that STAT3 regulates the transcription factor CCAAT / Enhancer Binding Protein,Delta(C/EBP?)activates UPS and causes muscle wasting.More importantly,skeletal muscle-specific knockout of STAT3 in mice,or application of STAT3 inhibitors,can significantly improve skeletal muscle atrophy in cancer cachexia mice.These studies suggest that STAT3 is an important target for the treatment of cancer cachexia.In this study,based on the pathological characteristics of cancer cachexia and the mechanism of skeletal muscle atrophy,we used STAT3 as the target and screened 1186 small molecule compounds that source from traditional Chinese medicine prescription of cachexia treatment based on surface plasmon resonance(SPR)analysis.The dual luciferase reporter gene analysis was used to verify the inhibitory effect of the compound on STAT3 transcription activity;the protective effect of candidate compounds on myotube atrophy was evaluated in vitro,and its effect on muscle wasting was studied in vivo.Finally,STAT3 gene overexpression technology was used to explore whether the candidate compounds exerted protective effects of skeletal muscle atrophy through STAT3 inhibition.This project can reveal the mechanism of candidate compounds improve cancer cachexia and also provide new ideas for the development of cancer cachexia treatment.Based on the above content,the research were carried out as followings:Purpose:1.Screen and find the active ingredients of traditional Chinese medicine that can bind and inhibit the activation of STAT3.2.To evaluate the active ingredients of Chinese medicine with high inhibitory activity of STAT3 to improve myotube atrophy and skeletal muscle atrophy in cancer cachexia.3.To explore whether the active ingredients of traditional Chinese medicine can improve skeletal muscle atrophy of cancer cachexia through STAT3 inhibition.Methods:1.SPR daul protein low-molecular-weight screening was used for screening 1186 small molecule compounds of traditional Chinese medicine.STAT3 protein and its control protein were immobilized on the surface of biosensor chip,and 1186 kinds of Chinese medicine small molecular compound solution of the same concentration were prepared to flow through the chip one by one.According to the difference of response signals between two proteins,the active ingredients of traditional Chinese medicine with relatively strong binding signal to STAT3 were screened.SPR Kinetics/Affinity analysis was used to verify the binding/dissociation process of active ingredients of traditional Chinese medicine with STAT3 protein at different concentrations,and the interaction mode and kinetic constant were analyzed.Then daul-luciferase reporter gene analysis was used to detect the effect of active ingredients of traditional Chinese medicine on IL-6+IL-6 receptor(Interleukin6 receptor,IL-6R)activated STAT3 transcriptional activity.2.Mouse C2C12 myotubes were used as the research object in vitro.Myotubes were treated with different concentrations of active ingredients of traditional Chinese medicine.The cell viability was detected by CCK-8 analysis to determine the concentration range of active ingredients.The myotube atrophy stimulated by the supernatant of CT26 colon adenocarcinoma cells(TCM)was used as an in vitro model.After the myotubes were treated with different concentrations of traditional Chinese medicine active ingredients,the expression of My HC protein and STAT3 nuclear translocation were detected by immunofluorescence,and the expression levels of p-STAT3,Mu RF1 and Atrogin-1proteins in myotubes were detected by Western blot.The protective effects of traditional Chinese medicine active ingredients on myotube atrophy and the expression of muscle degradation related proteins were analyzed.In vivo,CT26 colon adenocarcinoma was used to induce cancer cachexia in mice,and the animal models of pre-cachexia(22 days)and cachexia(30 days)were performed to detect the effects of the active ingredients of Chinese medicine on body weight,tumor growth,appetite,muscle and organ mass.Myofiber size and distribution were analyzed by HE staining.The protein expression levels of p-STAT3,Mu RF1 and Atrogin-1 in gastrocnemius muscle were detected by Western blot,and the expression levels of Murf1 and Atrogin-1 m RNA in gastrocnemius muscle were detected by Real-time q PCR.The treatment of the active ingredients of Chinese medicine on cancer cachexia and the expression of muscle degradation related proteins were analyzed in vivo.3.STAT3 overexpression lentivirus was constructed and infected with C2C12 myotubes.The expression of My HC protein in myotubes was detected by immunofluorescence,and the expression of p-STAT3,Mu RF1 and Atrogin-1 proteins were detected by Western blot.The effects of the active ingredients of Chinese medicine on myotube atrophy and protein degradation induced by TCM were analyzed in vitro.Lentivirus-mediated overexpression of STAT3 gene in gastrocnemius muscle was used to detect the effect of the active ingredients of Chinese medicine on skeletal muscle atrophy and muscle protein degradation in CT26-induced cancer cachexia.4.The CT26-induced cancer cachexia mice model was established,and the expression of p-STAT3 in tumor was analyzed by Western blot,the expression of p-STAT3 in tumor infiltrating lymphocytes was detected by flow analysis.To study the effect of the active ingredients of Chinese medicine on the activation of STAT3 in tumor and immune cells in vitro.To study the effect of the active ingredients of Chinese medicine on myotube atrophy and protein degradation induced by the co-cultured supernatant of tumor and immune cell,myotubes was stimulated by the supernatant of CT26,RAW264.7 and CT26 co-cultured with RAW264.7 cells,the expression of My HC protein in myotubes was detected by immunofluorescence,and the expression of p-STAT3,Mu RF1 and Atrogin-1 proteins were detected by Western blot.Results:1.The 10 active ingredients of traditional Chinese medicine in the small molecule compound library of traditional Chinese medicine,including 145,146,181,248,401,496,498,1034,1143,and 1186,can specifically bind to STAT3 protein and inhibit IL-6+ IL-6R activated STAT3 transcriptional activity in a dose-dependent manner.2.In vitro studies have shown that the active ingredients of traditional Chinese medicine imperatorin(181),saikosaponin D(496)and cryptotanshinone(1186)can dose-dependently protect TCM-induced myotube diameter reduction and inhibit the increased Mu RF1 and Atrogin-1 protein expression levels,and increased STAT3 phosphorylation levels and nuclear translocation.In vivo studies have shown that cryptotanshinone and imperatorin can significantly improve the weight loss and muscle wasting of CT26-induced cancer cachexia,and can dose-dependently inhibit p-STAT3,Mu RF1,Atrogin-1 protein and Murf1,Atrogin-1 m RNA expression levels in cachexia mice.However,mice treated with saikosaponin D showed an acute toxic reaction,and no further studies were conducted.Imperatorin has the best therapeutic effect on cancer cachexia and was selected to explore the mechanism.3.After overexpression of STAT3 in myotubes,the reversal effect of imperatorin on TCM-induced myotube atrophy and the expression of Mu RF1 and Atrogin-1 protein was greatly weakened,which proved that imperatorin could improve myotube atrophy by inhibiting STAT3.However,after overexpression of STAT3 in gastrocnemius muscle of mice,imperatorin can still reduce skeletal muscle atrophy and inhibit the expression of Mu RF1 and Atrogin-1 protein in CT26-induced cancer cachexia.Results show that imperatorin may also improve skeletal muscle atrophy through inhibiting STAT3 activation in tumor or immune cell.4.Imperatorin treatment not only inhibits tumor growth,but also inhibits the activation of STAT3 in tumor cells and tumor infiltrating lymphocytes,and reduces the expression levels of IL-6,TNF-? and IL-1 ? in serum and muscle.The co-culture supernatant of CT26 and RAW264.7 cells aggravated myotube atrophy,while the effect of myotube atrophy and the protein expression levels of p-STAT3,Mu RF1 and Atrogin-1 induced by co-culture supernatant of CT26 and RAW264.7 cells pretreated with imperatorin were significantly reduced.In addition,imperatorin can reduce the levels of IL-6 and TNF-? released by CT26 and RAW264.7 cells.Results showed that imperatorin could inhibit the STAT3 activation and reduce the release of pro-inflammatory factors in tumor and immune cells.Conclusion:The active ingredients of Chinese medicine imperatorin,saikosaponin D and cryptotanshinone can specifically bind to STAT3 and inhibit its transcriptional activity.In vivo and in vitro studies have shown that imperatorin has a strong therapeutic effect on TCM-induced C2C12 myotube atrophy and CT26-induced cancer cachexia.Further mechanism studies have shown that imperatorin directly inhibits the activation of STAT3 signal pathway in muscle,and inhibits the expression of downstream muscle protein degradation-related proteins,reduces muscle degradation and improves skeletal muscle atrophy.On the other hand,imperatorin indirectly acts on tumor and immune cells,inhibits STAT3 activation and the release of pro-inflammatory factors,improves muscle wasting and cancer cachexia.