Osthole Delays Hepatocellular Carcinoma Development Through Erk And Akt/mTORC1/FASN Pathways

Objective: Hepatocellular carcinoma(HCC)is one of the most common cancers in the world.About 200,000 people die of liver cancer every year in China.Despite the continuous development of therapeutics for HCC,the survival rate is still not high.Studies have shown that de novo fatty acid synthesis is an important cause of HCC occurrence and development,and that inhibition of the expression of the de novo fatty acid synthesis key enzyme fatty acid synthase(FASN)can inhibit the formation of HCC in mice.Traditional Chinese medicine is China’s traditional medicine,which plays an important role in many processes such as inhibiting tumor progression,reducing surgical complications,and improving the sensitivity of chemotherapy and radiation therapy.Therefore,it is a possible direction to look for compounds from Chinese herbal extracts to inhibit the expression of FASN to inhibit the abnormal synthesis of de novo fatty acids,thereby delaying the development of HCC.Osthole has various pharmacological effects such as anti-inflammatory,immunomodulation and anti-tumor,and studies in vitro and in nude mice have shown that it has certain therapeutic effect on HCC,but can it pass the AKT/m TORC1/FASN pathway? Regulating de novo fatty acid synthesis to inhibit the development of HCC has not been studied.The previous research results of this research group show that the use of tail vein high pressure transfection technology to simultaneously overexpress AKT and c-Met plasmids in mouse liver can establish a mouse HCC model,which has the characteristics of severe fatty and clear targets.This study intends to use this new HCC model to study the action mechanism of osthole through the Erk and AKT/m TORC1/FASN pathways to delay the development of HCC.Method:1.Twenty wild-type FVB/N mice were selected and randomly divided into a normal group,a model group,and a group of osthole low and high dose(120,240 mg·kg-1),five in each group.The model group and the low-dose and high-dose osthole group used SB-mediated tail vein high-pressure transfection technology to overexpress high-concentration AKT/c-Met plasmids in mouse livers to establish hepatocellular carcinoma models.Three weeks after modeling,the osthole low and high dose groups were injected intraperitoneally once a day to intervene;the model group and the normal group were given the same amount of blank solvent as a control;the weight of the mice in each group was recorded every 2 days.After 3 weeks of continuous drug intervention,the mouse venous blood was collected from the orbital venous plexus,centrifuged at 12000 r/min at 4 °C for 10 min,and the upper serum was stored in a refrigerator at-20 °C for storage;the complete mouse liver tissue was stripped and weighed for photograph Record,take a part of liver tissue and fix it,embed sections,and store the rest in-80 ° C refrigerator for future use.Statistical methods were used to compare the differences in body weight,liver weight,and liver index between mice in each group;HE staining was used to detect liver tissue lesions in each group;Elisa was used to detect the expression of tumor marker AFP protein in serum;q RT-PCR was used to detect liver The expression of tumor markers AFP and GPC3 m RNA in tissues.2.IHC was used to detect the expression of Ki67,PCNA and FASN protein in mouse liver tissues;Western blotting was used to detect the activation of proliferation-related protein Erk in mouse liver tissues and the key cascade of de novo fatty acid synthesis The expression levels of main proteins t-AKT,p-AKT T308,p-AKT S473,p-RPS6,and FASN in AKT/m TORC1/FASN pathway.3.Further use SMMC-7721 and Hep G2 two human hepatocellular carcinoma cell lines to study the possible mechanism of osteoblast on hepatocellular carcinoma.Transient transfection technology was used to transfect AKT/c-Met plasmid to Hep G2 with low AKT expression,then SMMC-7721 and Hep G2 cells after transfection with AKT/c-Met were given intervention of osthole.Western blotting is used to detect the activation of the proliferation-related protein Erk and the expression of the major proteins t-AKT,p-AKT T308,p-AKT S473,p-RPS6,and FASN in the de novo fatty acid key pathway AKT/m TORC1/FASNResult:1.Compared with the normal group,the mice in the model group weight,liver weight and organ index significantly increased;vacuolar degeneration,multi-nucleus and nuclear shrinkage of liver tissue were obvious;tumor markers AFP and The expression of GPC3 was significantly increased;compared with the model group,the mice in the low-and high-dose osthole groups body weight,liver weight and viscera index significantly decreased;vacuole degeneration,multi-nucleus,and nuclear shrinkage of liver tissues were reduced;nucleus Ki67 and PCNA positive staining decreased;the expression of tumor markers AFP and GPC3 in serum and liver tissues decreased significantly.2.Compared with the normal group,the results of IHC staining showed a large number of FASN positive staining in liver tissue;p-Erk1/2 and p-AKT T308,p-AKT S473,p-RPS6,FASN protein in liver tissue of model group Expression increased significantly.FASN showed the same IHC and Western blotting results.The above indexes were significantly improved after osthole intervention and showed a dose-effect relationship.3.The expressions of p-Erk1/2 and p-AKT T308,p-AKT S473,p-RPS6,and FASN in SMMC-7721 and Hep G2 cells transfected with AKT/c-Met plasmid were significantly reduced after intervention with osthole.Consistent with results from in vivo studies.Conclusion: Osthole can delay the development of hepatocellular carcinoma induced by AKT/c-Met in mice,and its mechanism of action may be to inhibit Erk activation and AKT/m TORC1/FASN signaling pathway,thereby preventing the abnormal proliferation of hepatocellular carcinoma.