The Mechanism Of Single-walled Carbon Nanohorns Motivating Mitochondrial Dysfunction Induced Apoptosis In HepG2 Cells

Background and objectiveHepatocellular carcinoma(HCC)is a common malignant tumor of the digestive system characterized by high drug resistance and lack of radical treatment.Most patients have metastasized at the time of the visit.It is difficult to completely eliminate the cancer in the body.In addition,the early diagnosis of HCC is difficult,and it is worthwhile to study new cytotoxic drugs and substances targeting specific molecular targets[1].Single-walled carbon nanohorns(SWNHs),a novel nanocarbon that does not contain any metal catalysts,are receiving increasing attention due to their unique morphology and structure[2].It has been shown that SWNHs can inhibit the growth,proliferation and mitosis of normal human liver cells and cancer cells,and promote apoptosis[3].However,there is a lack of further exploration of its upstream molecular mechanisms and effects on subcellular structures.Objective1.To study the corresponding changes in the mitochondrial apoptosis pathway-related proteins in the process of liver cirrhosis to liver cancer.2.To investigate the effects of SWNHs on mitochondrial membrane potential,Na+-K+-ATPase activity and mitochondrial apoptosis pathway-related proteins in HepG2 cells and L02 cells in vitro.3.To investigate the effects of SWNHs on mitochondrial apoptosis pathway-related proteins in nude mice and in vivo HepG2 cells.Experiment:1.Collect liver cancer,paracancerous,and liver cirrhosis tissue samples from liver cancer patients,and detect the expression of mitochondrial apoptosis-related proteins by immunohistochemistry.2.HepG2 and L02 cells were treated with different concentrations of SWHNs,and the mitochondrial membrane potential detection kit was used to detect the effect of SWNHs on cell mitochondrial membrane potential.The Na+-K+-ATPase assay kit was used to detect the effects of different concentrations of SWNHs on Na+-K+-ATPase activity in HepG2 cells.Western blotting was used to detect the effect of SWNHs on the expression of mitochondrial apoptosis-related proteins in HepG2 cells and L02 cells.3.Construct HepG2 cells-nude mouse xenograft model,and regularly treat different concentrations of SWNHs in the tumor to observe the volume change of the tumor and the weight and nutrition of the nude mice.Hematoxylin-eosin(HE)staining was used to observe the morphological changes of HepG2 cells in the tumor.The distribution of fibrin in the nodules was observed by Sirius red staining,and the expression of mitochondrial apoptosis-related proteins was detected by Western blotting.Immunohistochemistry was used to further detect mitochondrial apoptosis-related protein expression levels and intracellular localization.Result:1.The difference between the positive intensity of SIRT3 and CYT-C in tumor tissues and the paracancerous or cirrhosis was statistically significant,and the expression levels of the two proteins were significantly decreased.The expression of AceCS2 in liver cancer tissues showed an upward trend.There was no significant difference in the expression level and expression position between Bax,SCNNla and VDAC1 in cirrhosis,adjacent tissues and cancer tissues.2.SWNHs can induce mitochondrial membrane potential depolarization in HepG2 cells,which has little effect on mitochondrial membrane potential of L02 cells.Na+-K+-ATPase assay indicated that SWNHs could inhibit Na+-K+-ATPase activity.The results of Western blotting showed that SWNHs up-regulated the expression of SIRT3,VDAC1 and CYT-C,and down-regulated the expression of AceCS2 and Bax,but had little effect on SCNN1a.However,SWNHs had little effect on L02 cell mitochondrial apoptosis pathway protein.3.Compared with the control group,SWNHs had no significant difference in body weight,nutritional status,and gross volume,cell morphology,number of apoptotic bodies,and interstitial fibrous tissue distribution of HepG2 cells.The results of Western blotting indicated that SWNHs could up-regulate the expression of SIRT3 and Bax in HepG2 xenografts,but had little effect on VDAC1,AceCS2,CYT-C and SCNNla.The results of immunohistochemistry indicated that SWNHs significantly up-regulated the expression of SIRT3,VDAC1 and Bax in HepG2 xenografts,and significantly down-regulated the expression of AceCS2.There was no statistical difference in the effect of CYT-C.In addition,after treatment with SWNHs.SIRT3 and SCNNla showed co-expression of nucleus and cytoplasm;AceCS2 cytoplasm had less expression,but the expression level of nucleus increased,while the expression of Bax,VDAC1 and CYT-C did not change much..Conclusion:During the process of carcinogenesis of cirrhosis,the mitochondrial apoptosis pathway protein is inactivated,the expression of apoptosis protein in the nucleus is reduced,and the intracellular mitochondrial capacity is active.SWNHs can promote mitochondrial membrane potential depolarization,inhibit ATPase activity,mitochondrial dysfunction,promote the overexpression of mitochondrial apoptosis pathway proteins(such as SIRT3,CYT-C,etc.)and release into the cytoplasm,combined with cytosolic apoptotic factors.Inducing apoptosis in liver cancer cells.