保元解毒汤通过P38MAPK/PGC-1通路调控线粒体功能改善癌性恶病质肌肉萎缩机制研究

The preservation of muscle weight is the key to survival in patients with cancer cachexia. Previous studies indicate that Baoyuan Jiedu Decoction’s function of improving the muscle atrophy is related to biological oxidation. It is hypothesized that Baoyuan Jiedu Decoction improves the muscle atrophy by reducing the inflammatory cytokine content, activating P38MAPK/PGC-1 signal pathway, regulating mitochondrial function. A mouse model of Apcmin/+ is used in vivo to observe the effect of Baoyuan Jiedu Decoction on body weight, muscle weight and intestinal adenomas in different development stages of cachexia dynamically; fluorescent staining, Real-PCR and Western blot, etc. are used to detect inflammatory cytokine, observe mitochondrial morphology, and determine mtDNA content, mitochondrial function related factors NRFs, TFAM, Cytochrome C, UCPs, Mfns, etc., P38MAPK/PGC-1 pathway atrophy, the protein and mRNA expression of mitochondria-related FoxOs and MuRF-1. A cancer-related myotube atrophy model is established in vitro to study the change of pathway and mitochondrial function after the intervention of serum contained surge and channel antagonist. It will provide a scientific illustration of the mechanism of Baoyuan Jiedu Decoction improving the muscle atrophy by regulating mitochondriopoiesis, oxidative phosphorylation function, uncoupling, polymerization and lytic function via P38MAPK/PGC-1 Pathway.

保存肌肉重量是癌性恶病质患者生存的关键。前期研究提示保元解毒汤改善肌肉萎缩与线粒体生物氧化相关。提出保元解毒汤通过降低炎性细胞因子含量，激活P38MAPK/PGC-1信号通路，调控线粒体功能，改善肌肉萎缩的假说。采用Apcmin/+小鼠模型，动态观察本方对恶病质不同进展期生存质量的影响，运用荧光染色、RTQ-PCR、Western blot等方法，检测炎性细胞因子，观察线粒体形态，测定mtDNA含量，NRFs、TFAM、Cytochrome C、UCPs、Mfns等线粒体功能相关因子，P38MAPK/PGC-1通路分子，萎缩相关FoxOs、MuRF-1等蛋白及mRNA表达；体外建立癌因性肌管萎缩模型，含药血清及通路拮抗剂处理，研究通路分子及线粒体功能改变。科学阐释保元解毒汤干预P38MAPK/PGC-1信号通路，调控线粒体生成、氧化磷酸化功能、解偶联作用和聚合裂解功能，改善肌肉萎缩的机制。

目的：以P38MAPK/PGC-1通路为切入点，研究保元解毒汤调控线粒体功能改善癌因性肌肉萎缩的机制。方法：HPLC法检测保元解毒汤中绿原酸、阿魏酸和乌头碱含量，评价其稳定性。体内建立C26移植瘤癌性恶病质模型。实验分为5组：正常对照组、模型组、中药组、甲地孕酮组和P38MAPK拮抗剂组。进行生活质量评价，测定肌肉中Atrogin-l、MuRF-1、P38MAPK及PGC-1 mRNA和蛋白表达。体外建立Lewis细胞条件培养下小鼠成肌细胞（C2C12）肌管萎缩模型。实验分为7组：正常对照组、模型组、中药组、P38MAPK拮抗剂组、中药+P38MAPK拮抗剂组、PGC-1α激动剂组和中药+PGC-1α激动剂组。观察保元解毒汤干预后肌管形态及线粒体功能变化，检测mtDNA拷贝数、ATP酶活性，检测线粒体功能相关Myf6、MyoD、MYH3、TFAM、NRF-1、CytC、COXIV以及P38MAPK/PGC-1α 通路相关蛋白及mRNA表达，探讨保元解毒汤调控线粒体功能的机制。结果：HPLC结果显示，保元解毒汤中绿原酸含量约114.3~114.5 ug/mL，阿魏酸含量约65.89~66.01 ug/mL，乌头碱含量约14.60~14.76 ug/mL，表明保元解毒汤质量稳定。体内：与模型组比较，保元解毒汤能显著缓解体质量下降（P<0.05），显著增加肌纤维横径及腓肠肌重量（P<0.05），显著降低Atrogin-l、MuRF-1、P38MAPK及上调PGC-1α mRNA及蛋白表达水平（P<0.05）。体外：与正常组比较，模型组中C2C12细胞肌管萌发延迟且数量明显减少，肌管横径显著下降（P＜0.05）。与1:4和1:8浓度相比，1:2浓度的条件培养基组中C2C12肌管数量及横径显著下降（P＜0.05）。与模型组相比，保元解毒汤明显增加肌管横径（P＜0.05），同时显著增加线粒体数量、ATP合成酶及mtDNA拷贝数（P<0.05），显著上调NRF1、TFAM、COXIV、CytC、MyoD、Myf6和MYH3 mRNA和蛋白表达（P＜0.05），明显下调p-P38MAPK蛋白和mRNA表达（P＜0.05）及上调PGC-1α mRNA表达。结论：保元解毒汤可通过P38MAPK/PGC-1通路改善线粒体功能进而减轻癌性恶病质。