Mechanism Of Ginsenoside Rg3 Improving Cisplatin-Induced Muscle Atrophy Of Ovarian Cancer Cachexia

Background and Objectives:Cancer cachexia(CC)is a multifactorial metabolic syndrome that can cause up to 40% of cancer-related deaths.Among them,muscle atrophy is a key feature of this syndrome.Ovarian cancer is the most deadly gynaecological malignancy.As a commonly used first-line chemotherapy drug for ovarian cancer,cisplatin can cause cachexia symptoms such as muscle atrophy,loss of appetite,and weight loss.Oxidative stress is believed to be closely related to muscle atrophy in cancer and cachexia caused by chemotherapy.It can directly or indirectly cause muscle atrophy through redox signals in the degradation pathway,and using antioxidants can effectively alleviate it.20(R)-Ginsenoside Rg3(20(R)-Rg3)is the main active ingredient isolated from red ginseng(Ginseng C.A Meyer),which has been proven to have antioxidant effects and is widely used in clinical practice as an adjuvant anti-tumour therapy.However,its role and mechanism in cancer cachexia and muscle atrophy are still unclear.This study explored whether ginsenoside Rg3 can improve cancer cachexia and muscle atrophy through its antioxidant effect at the animal and cell levels,providing a theoretical basis for the treatment and application of ginsenoside Rg3 in cachexia and muscle atrophy.Methods:1.C57BL/6J mice were injected intraperitoneally with ID8 mouse ovarian cancer cells to establish an animal model of cancer cachexia.Normal saline,cisplatin intraperitoneal perfusion chemotherapy,or cisplatin intraperitoneal perfusion chemotherapy combined with Rg3 intragastric treatment was administered to observe the changes in body weight,food intake,gastrocnemius muscle,quadriceps femoris muscle,heart,liver,spleen,kidney weight,and tumour growth in different treatment groups.2.HE staining of gastrocnemius muscle fibre cross-section,Image J analysis of the average fibre cross-sectional area and frequency distribution of large and small muscle fibres in each group of mice gastrocnemius muscle;Immunohistochemistry detected the expression of myosin heavy chain type I fibres(My HCI)in the gastrocnemius muscle of mice.Image J analyzed the proportion changes of myosin heavy chain type I fibres(My HCI)and myosin heavy chain type Ⅱ fibres(My HCⅡ);The mRNA levels of type Ⅰ,Ⅱa,and Ⅱb muscle fibre marker genes MYH7,MYH2,and MYH4 in mouse gastrocnemius muscle were detected by qPCR.3.Malondialdehyde(MDA)and superoxide dismutase(SOD)kits were used to detect the levels of MDA and SOD in the gastrocnemius muscle of mice in each group;Western blot detected the AMPK/SIRT1/PGC-1α signal pathway expression in gastrocnemius muscle of mice in each group.4.C2C12 myoblasts were induced to differentiate into myotubes using high glucose DMEM medium containing 2% horse serum.The model of C2C12 myotube atrophy was established by adding conditioned medium containing cisplatin and different concentrations of Rg3 or adding cisplatin alone to ID8 mouse ovarian cancer cells to simulate the tumour microenvironment in vivo.Crystal violet staining was used to observe the morphological changes of C2C12 myotubes,and Image J was used for quantitative analysis of myotube width;The mRNA levels of muscle-specific ubiquitin protein ligase 1(Mu RF1)and muscle atrophy protein Fbox-1(Atrogin-1)were detected by qPCR.5.After treatment with AMPK inhibitor Compound C or SIRT1 inhibitor EX527,Western blot was used to detect the protein expression of AMPK/SIRT1/PGC-1α signal pathways and Mu RF1 and Atrogin-1 in C2C12 myotubes induced by ID8 mouse ovarian cancer conditioned medium supplemented with cisplatin and Rg3 or cisplatin alone.Results:1.Rg3 can alleviate weight loss,reduced food intake,and weight loss of gastrocnemius muscle,quadriceps femoris muscle,heart,liver,and kidney in ID8 ovarian cancer mice caused by intraperitoneal infusion chemotherapy with cisplatin without affecting the antitumor activity of cisplatin.2.Rg3 can alleviate the decrease in the cross-sectional area of skeletal muscle fibres in ID8 ovarian cancer mice undergoing intraperitoneal infusion chemotherapy with cisplatin,improve the size distribution of the cross-sectional area of muscle fibres,and change the proportion of fibre types,especially maintaining the proportion of type Ⅱ muscle fibres.3.Rg3 can alleviate the increase of MDA level and decrease SOD content in skeletal muscle of ID8 ovarian cancer mice treated with intraperitoneal perfusion chemotherapy with cisplatin and activate AMPK/SIRT1/PGC-1α signal pathway.4.Rg3 can alleviate C2C12 myotube atrophy induced by adding cisplatin-containing ovarian cancer conditioned medium to ID8 mice and downregulate the mRNA levels of myotube atrophy specific related proteins Mu RF1 and Atrogin-1.5.Rg3 can activate AMPK/SIRT1/PGC-1α pathway alleviates C2C12 myotube atrophy induced by adding cisplatin-containing ovarian cancer-conditioned medium in ID8 mice,while AMPK inhibitor Compound C and SIRT1 inhibitor EX527 reverse the protective effect of Rg3.Conclusions:1.Rg3 improved cachexia symptoms and oxidative stress in the skeletal muscle of ID8 ovarian cancer mice undergoing intraperitoneal infusion chemotherapy with cisplatin and does not affect the antitumor activity of cisplatin.2.Rg3 activated AMPK/SIRT1/PGC-1α signal pathways to alleviate muscle atrophy in cancer cachexia.