Mechanism Of Baoyuan Jiedu Decoction On Intervening The Cancerinduced Muscle Atrophy By Regulating Mitochondrial Function Based On P38MAPK/PGC-1 Pathway

Objective: To observe the effects of Baoyuan Jiedu Decoction on cancer-induced muscle wasting through regulating the mitochondrial function based on P38MAPK/PGC-1 pathway.To find a poossible target of Baoyuan Jiedu Decoction on cancer-induced muscle wasting,and provide experimental evidence for the development of effective Chinese medicine for cancerinduced muscle wasting.Method:1 We used HPLC method to qualitative and quantitative analysize the active ingredients chlorogenic acid,ferulic acid and aconitine in Baoyuan Jiedu Decoction(BYJD)to ensure the material basis of the effect of Baoyuan Jiedu Decoction;The mice were treated with BYJD for 10 days after the C26 cachexia model were established.We observed the general condition and body weight of the animals,and detected the gastrocnemius muscle weight and stained the muscle with HE to evaluate the intervention of BYJD on cachexia animal muscle atrophy;meanwhlie,we detected the expression of P38 MAPK,PGC-1α,Atrogin-1 and Mu RF-1m RNA and protein by RT-PCR and WB to evaluate the effect of BYJD on C26 cachexia model mice and explore the mechanism;2 Establishment and evaluation of cancer-induced muscle wasting model.We cultured C2C12 cells with different conditioned medium contained with 1:2,1:4,1:8 concentrations of Lewis cell supernatant for 96 h,and observed the changes of myotubes in C2C12 cells by ordinary inverted microscope,then detected the level of TNF-α and IL-6 by ELISA to constructe a model of cancer-induced muscle wasting;3 Study on the effect and mechanism of BYJD on alleviating cancer-induced muslce wasting:3.1 We added BYJD to the culture medium at a ratio of 1:8,1:16 and 1:32 to intervene C2C12 cells for 72 h.We checked the changes of myotubes in C2C12 cells with ordinary inverted microscope,detected the contents of TNF-α and IL-6 by ELISA,and detected the expression of Myf6,MYH3,Myo D,Atrogin-1 and Mu RF-1 m RNA and protein by RT-PCR and WB to find out the best intervention time and concentration,and evaluate the effect of BYJD on cancerinduced muscle wastiong;3.2 After the cancer-induced muscle wasting model cells were intevened by the appropriate concentration of BYJD,SB203580 and ZLN005 for 72 h,we observed the myotubes changes in C2C12 cells using ordinary inverted microscope,and detected the the expression of P38 MAPK,PGC-1α m RNA and protein by RT-PCR and WB to evaluated the effect of BYJD on P38MAPK/PGC-1 pathway;3.3 We also observed the mitochondria after NAO staining to evaluate the effect of BYJD on mitochondria number;detected the mt DNA number by RT-PCR and detected the expression of NRF1,TFAM,m RNA and protein by RT-PCR and WB to evaluate the effect of BYJD on promoting mitochondrial biosynthesis;then we checked the the ATPase activity by biochemical method,detected the expression of ATP synthaseβ subunit by RT-PCR,and explored the expression of COXIV,cytochrome C by RT-PCR and WB to evaluate the mechanism of BYJD on mitochondrial fuction.Results:1 According to HPLC,the content of chlorogenic acid in Baoyuan Jiedu Decoction was about 114.3~114.5 ug/m L,the content of ferulic acid was about 65.89~66.01 ug/m L,and the content of aconitine was about 14.60~14.76 ug/m L,and the three substances are stable in BYJD;Compared with the model group,BYJD effectively inhibited the body weight loss,increased the muscle weight and the transverse diameter of gastrocnemius muscle;and inhibited the P38MAP/PGC-1α pathway,reduced the expression of Atrogin-1 and Mu RF-1;2 The cancer-induced muscle wasting model of C2C12 was successfully established.After C2C12 cells were cultured with different concentrations of Lewis lung adenocarcinoma cells supernant,Myotubes germination was delayed and the number was significantly reduced and the transverse diameter of myotubes was significantly thinner than the normal,and these effects are all positively correlated with the duration of action and concentration.There was a statistically significant difference in the transverse diameter of C2C12 cells between the groups(P<0.05),the best intervention time was 96 h and the optimum concentration was 1:2;3 BYJD alleviated cancer-induced muscle wasting,blocked the P38MAPK/PGC-1 pathway,increased the number of mitochondria and improved the function of mitochondrial:3.1 The transverse diameter of the myotube were increased treated with 1:32,1:16,1:8 concentration of BYJD with dose and time-dependent manner(P<0.05).The effect of 1:8 concentration intervented for 72 h was best of all group.The expression of Mu RF-1 and Atrogin-1 m RNA and protein were significantly lower than that of model group after treatment with 1:8 BYJD for 72h(P<0.05),while the m RNA and protein expression of Myo D,Myf6 and MYH3 were increased compared with the model group(P<0.05);3.2 Compared with the model group,BYJD inhibited P38 MAP m RNA expression and protein phosphorylation,promoted PGC-1α m RNA and protein expression(P<0.05);3.3 Compared with the model group,BYJD increased the number of mitochondria in C2C12 cells and mt DNA copy number(P<0.05)dramatically,increased the expression of NRF1 and TFAM(P<0.05);increased the level of ATP synthase β subunit(P<0.05),up-regulated ATPase activity,promoted COXIV and Cytc expression(P<0.05).Conclusion:The results of in vivo study showed that BYJD could relieve muscle wasting by inhibiting P38MAPK/PGC-1 pathway and inhibiting the expression of muscle atrophy-associated protein;Meanwhlie,the results of in vitro study have shown that the mechanism of BYJD on alleviating cancer-induced muscle atrophy may be related to reducing cytokine expression,inhibiting P38MAPK/PGC-1 pathway,promoting mitochondrial production,and improving mitochondrial oxidative phosphorylation.