Mechanism Of Ginsenoside Rg1 Regulating Nonalcoholic Fatty Liver(NAFLD)via Sphingosine Kinase Pathway

Non-alcoholic fatty liver disease(NAFLD)refers to a substantial pathological change in the liver caused by excessive alcohol intake or genetic susceptibility,which can further develop into non-alcoholic fatty liver disease(NASH),liver cirrhosis,hepatocellular carcinoma,liver failure.Studies have shown that NAFLD is also closely related to various metabolic diseases,such as central obesity,chronic kidney disease(CKD),type 2 diabetes(T2DM),hypertension,and cardiovascular disease.At present,insufficient attention has been paid to NAFLD clinically,and no consensus has been reached on its pathogenesis,diagnosis,prevention and treatment.In recent years,studies have shown that apoptosis may be involved in the pathogenesis of NAFLD.Apoptosis is a method of programmed cell death,including a series of changes in cell morphology and function.The causes of NAFLD are complex,including disorders of lipid metabolism,insulin resistance and oxidative stress,and apoptosis and inflammation caused by endoplasmic reticulum stress.Sphingosine kinase pathway is a signaling pathway based on lipid metabolism and regulating cell survival and apoptosis,and plays an important role in NAFLD-related liver fibrosis.In the treatment and prevention of NAFLD,there are few reports on the research and clinical experiments on regulating lipid metabolism and interfering with apoptosis.Therefore,the relationship between lipid metabolism and apoptosis in the sphingosine kinase pathway is urgently needed to provide the necessary theoretical basis for the development of related drugs.So far,there are no clear specific drugs to treat NAFLD clinically.Therefore,finding drugs that can regulate fat metabolism and apoptosis has become a hot spot in medical research.Ginsenoside Rg1 is a Tetracyclic triterpenes Object extracted from panax notoginseng and ginseng,and is the main active substance in ginseng plants.Previous studies by some scholars have shown that ginsenoside Rg1 can reduce lipid deposition,alleviate oxidative stress,inhibit inflammatory responses and resist apoptosis of liver cells,and protect liver function.Based on the relevant literature reports and the previous work of the research group,we made the scientific hypothesis that ginsenoside Rg1 may regulate lipid metabolism through sphingosine kinase pathway,affect cell survival and apoptosis,and then alleviate the pathogenesis of NAFLD,but the specific mechanism remains to be determined.Validated by us.In this experiment,a NAFLD cell model was established,and then Rg1 was administered at different concentrations,and then oil red O staining was performed to determine cell viability.Flow cytometry was used to detect apoptosis,and to investigate changes in m RNA and protein expression levels of sphingosine kinase pathway molecules.;And up-regulate SGPL,a key gene of the sphingosine kinase pathway,that is,infect the target cells with lentivirus,select stable transfected cell lines,perform cell modeling again,determine the expression of related molecules and phenotypic determination to verify our hypothesis and guess.This topic is mainly carried out from the following aspects:(1)Establishment of NAFLD cell model and drug treatment: The medium and long chain fat emulsion commonly used in clinical practice HHL-5 hepatocytes were induced to simulate a high-fat environment;then ginsenoside Rg1 was administered for treatment and identified with oil red O staining.The results showed that the cells were tightly arranged and polygonal;purple-red lipid droplets appeared in the cell after oil red O staining,and were located at the edge of the cell membrane ring;cell lipid droplets were reduced and cell morphology was clear after Rg1 treatment.(2)The effect of ginsenoside Rg1 on the proliferation and viability of HHL-5cells was tested using MTS experiments.The results showed that the Rg1 concentration explored in this subject at 0.2 m M,0.4m M,and 0.6 m M had no significant effect on cell proliferation.(3)The effects of ginsenoside Rg1 on cell cycle changes and apoptosis of HHL-5 cells were detected by flow cytometry.The results showed that the apoptosis of Rg1 was alleviated after the intervention of Rg1,and the apoptosis rate gradually decreased with the increase of Rg1 concentration.At the same time,Rg1 had no significant effect on HHL-5 cell cycle.(4)HHL-5 cells were treated with ginsenoside Rg1,and the expression of sphingosine kinase pathway related proteins was detected by RT-PCR and Immuno blot.The results showed that Rg1 inhibited the expression of apoptosis-regulating proteins BAX,Caspase-3,and SGPL-1;up-regulated BCL-2 and expression,and showed a dose-dependent relationship.(5)Upregulate SGPL to establish HHL-5 stably transfected cell lines,and then use Western to detect the expression of sphingosine kinase pathway related proteins.The results showed that after overexpression of sphingosine kinase pathway SGPL,the above phenotypic changes were not obvious after intervention with Rg1.This suggests that our conjecture is correct,and Rescue’s rescue experiment also confirmed this.Conclusions: Through the above experiments,we believe that: 1.NAFLD cell model can be successfully established by intravenous injection of fat emulsion in medical use,and it is relatively simple and practical.2.Ginsenoside Rg1 had no significant effect on the proliferation of NAFLD model cells.3.Ginsenoside Rg1 inhibits apoptosis.The mechanism may be related to the down-regulation of the sphingosine kinase pathway pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2.4.The viability of cells stably transformed after lentiviral packaging is lower than that of normal cells.It may be that over-expressed SGPL1 is not conducive to cell growth.5.The anti-apoptotic effect of ginsenoside Rg1 on over-expressed cells is not obvious,that is,our rescue of Rescue phenotype confirms that Rg1 indeed regulates the cell phenotype of NAFLD through the sphingosine kinase pathway.