| ObjectiveTo establish a stable method of quality control standard of CGTF,and estabilsh a model of alcohol-induced hepatocute steatosis in L 02 cells in vitro,investigate the function of CGTF in cellular and molecular levels to preliminarily study the effect and mechanism of an anti-alcoholic fatty liver of CGTF,which will provide a theoretical basis for its function.Methods1 Prepare CGTF by water extraction and alcohol precipitation,detemine the content of CGTF by spectrophotometer,examine the narringin by UPLC,and compare the printerfingers of CGTF from different varieties.Then,integrate the above data to establish the quality control standard of CGTF.2 The best ethanol concentration and the range of CGTF concentration to induce liver L 02 cells teatosis and CGTF were established by MTT method.Then observed the morphology of the control group?the model group and the CGTF groups of different concentrations by light inverted microscope,examined the content of LDH,detected the apoptosis rate by AnnexinV/PI.Then L02 cells were stained with red O-oil and observed by light inverted microscope.AST?ALT?TG?TC?SOD?GSH?MDA were analyzed with AST-kit?ALT-kit?TG-kit?TC-kit?SOD-kit?GSH-kit?MDA-kit,ROS was detected by the fluorescence spectrophotometer detection.And the protein of PPAR-??SREBP-1c were detected by western blot.Results1 Quality control standards of CGTFThe water extraction and alcohol precipitation method was simple and suitable for industrial product.CGTF was determined by Spectrophotometer and it'was simple in operation.The extraction rate was above 80%;we determined CGTF by UPLC,it showed naringin accounted between 73.16%and 80.09%;we compared the frinterprints of CGTF for 11 batches from different variety,the results showed that we got 15 common peaks with consistent reservation time,RSD was less than 0.2%,while peak area had big difference,but the sum of area of common peak was over 99%,the 8th peak was naringin,the 10th peak was rhoifolin,the 11th peak was naringing.2 The study of CGTF on ethanol-induced fatty degeneration in L 02 cell and its metabolism2.1 The MTT study showed that 0.3%(V/V)alcohol can promote proliferation,the cell survival rate was 80%when the ethanol concentration was 0.6%,but it reduced as the ethanol concentration and the effecting time increased.So 0.6%was selected as the most optimum concentration.The MTT study shows that CGTF can promote proliferation when its concentration was less than 250?g/mL,but it did't show regularity;when over 250?g/mL,it precipitated crystallization,when it reached 1000?g/mL,lots of crystallization was precipitated,but the cell survival rate of L 02 was still over 70%after cultured for 48 hours.2.2 With the cell morphology observtion,the number of cells of the model group was slightly less than the blank group and the cell mentality was slightly swelling,and the cells of CGTF group were normal.The results of LDH showed that 0.6%alcohol can damage the L02 cell,which was statistically significant(P<0.05),and when 100?g/mL or 250?g/mL affectted on the model group,the results were statistically significant(P<0.05).But compared with the blank group,Annexin-?/PI apoptosis experiments showed the model group wasn't statistically significant(P>0.05),and the apoptosis rate of CGTF groups didn't reduce(P>0.05).2.3 After incubated in 0.6%alcohol for 24 hours,numerous lipid droplets were scattered in the cytolymph of human L-02 hepatocyte by inverted microscope,which can be dyed to red with oil red O dying.After exposed to CGTF(10?g/mL?50?g/mL?100?g/mL and 250?g/m)for 24 hours,the intracellular lipid droplets of steatosis hepatocytes were much less than the model group by oil red ostain.And AST?ALT in the culture medium were found out to be increased dramatically through measurement of microplate reader(P<0.01),when the concentration was 10?g/mL,the difference was statistically significant(P>0.05),when more than 10?g/mL,the different was particularly statistically significant(P<0.01).The intracellular TG levels of the model group was statistically significant(P<0.05),when the concentration of CGTF was 10?g/mL,the difference was statistically significant(P<0.05),when more than 10?g/mL,the difference was particularly statistically significant(P<0.01).Compared with the blank group,the intracellular TC levels of the model group was statistically significant(P<0.05),when the concentration was 10?g/mL,the difference was statistically significant(P<0.05),when more than 10?g/mL,the different was particularly statistically significant(P<0.01).Thus,the model of steatosis hepatocytes was established successfully,and the AST?ALT?TG?TC levels decreased remarkably with dose-dependent pattern.2.4 After incubated in 0.6%alcohol for 24 hours,the intracellular GSH?SOD levels were found out to be decreased dramatically through measurement of microplate reader.the difference of SOD is statistically significant(P<0.05)and GSH(P<0.01),when the concentration of CGTF was more than 100?g/mL,the difference was statistically significant(P<0.05).The difference of MDA was statistically significant(P<0.01),when the concentration of CGTF was more than 100?g/mL,the difference was statistically significant(P<0.01).The difference of ROS was statistically significant(P<0.01),when the concentration of CGTF was more than 50?g/mL,the difference was statistically significant(P<0.01).After exposed to CGTF with different concentrations for 24 hours,the intracellular GSH?SOD levels were much less than the model group,the MDA?ROS levels decreased remarkably with dose-dependent pattern.2.5 Western blot was employed to detect the content of the protein of SREBP-lc and PPAR-? in the hepatocytes of each group,when more than 10?g/mL,the expression of PPAR-? was found out to be much higher in the group treated with CGTF than in the model ones,which was statistically significant(P<0.01),but it wasn't statistically significant(P>0.05)in the model ones compared with the blank group.The expression of SREBP-1c was found out to be much lower in the group treated with CGTF than in the model ones,which was statistically significant(P<0.01).But,compared with the control group,the expression of SREBP-1c was found out to be lower,when the concentration of CGTF was 10?g/mL,when more than 10?g/mL,the difference was statistically significant(P<0.01).Conclusion1 The CGTF was light yellow or yellow-green powder,and bitter.Flavonoids content was not less than 80%,naringin content was not less than 73%,the HPLC fingerprint of different varieties had a totel of 15 common peaks with the same retention time,and the totel area was not less than 99%,the similarity was above 0.98.2 CGTF can protect the L02 cell induced by alcohol in vitro by anti-cell damage?anti-oxidative stress and regulate protein content involved in fat metabolism.The experiment have successfully established alcoholic fatty liver model by L 02 cells induced by 0.6%(V/V)alcohol in vitro.The modeling methods was simple and drives short,by which we can directly observe the drug effects and the mechanism.The study was about the effect and pharmacological mechanism of CGTF on alcoholic fatty liver model in cellular and molecular levels.We found that CGTF can decrease the content of AST?ALT?TG?TC.The study shows the content of LDH increases when 0.6%alcohol affect on L02 cells,and CGTF can make it lower,it shows that CGTF can protect the cells from damage.But the apoptosis rate have never been affected by 0.6%alcohol or CGTF.0.6%alcohol can enhance oxidative stress,increase the content of MDA and ROS,decrease the content of SOD and GSH,and CGTF can decrease the content of MDA and ROS,increase the content of SOD and GSH.CGTF can regulate fat metabolism of steatosis hepatocyte to make the fat metabolism back to normal by activating the cyclin of PPAR-a and inhibiting the cyclin of SREBP-lc with a dose dependent manner. |
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