| Paper one: Effects of Huayuqutan Recipe on lipid deposition and autophagy in Hep G2 cells induced by FFAPurpose:Serum pharmacological methods were used to observe the effects of Huayuqutan Recipe on FFA-induced lipid deposition and autophagy in Hep G2 cells,and to explore the possible mechanism of Huayuqutan Recipe on preventing and treating hyperlipidemia.Materials and Methods:Hep G2 cells were induced to culture for 24 h with a 2:1 molar ratio of oleic acid and palmitic acid.Using CCK-8,oil red O staining and photoelectric colorimetric method to select the optimal concentration of Hep G2 cells to establish the induced lipid concentration model;CCK-8 method was used to determine the degree of drug-containing serum in rats.The experiment was divided into 4 groups: blank control group,model group,Huayuqutan Recipe group and simvastatin group.The effects of Huayuqutan Recipe on lipid deposition in Hep G2 cells induced by FFA were observed by oil red O staining and photoelectric colorimetry.Western Blot method was used to detect the effect of Huayuqutan Recipe on Atg3 and Atg5.Result:1.CCK-8 assay showed that the inhibition of Hep G2 cell viability was greater when the final concentration of oleic acid and palmitic acid mixture was more than 1 mmol/L after 24 h induction.2.Compared with the fetal calf blank serum group,after serum concentrations of 5%,10%,and 15% rat serum,Huayuqutan Recipe-containing serum,and simvastatin-containing serum were cultured for 48 hours,the concentration of 10% drug-containing serum had the smallest effect on cell viability.3.The oil red O method showed that compared with the blank control group,the formation of lipid droplets in the model group increased significantly;Compared with the model group,the formation of lipid droplets in the simvastatin group and Huayuqutan Recipe group was significantly reduced.4.Electro-optical colorimetry showed that compared with the blank control group,the intracellular TG content in the model group increased significantly(P<0.05);Compared with the model group,the intracellular TG content of the simvastatin group and the Huayuqutan Recipe group was significantly decreased(P<0.05).5.Western Blot showed that 3h: compared with the model group,the expression of Atg3 and Atg5 in the Huayuqutan Recipe group was significantly increased(P<0.05);6h: Compared with the blank control group,the expression of Atg3 and Atg5 in the Hep G2 cells of the model group increased significantly(P<0.05);Compared with the model group,the expression of Atg3 and Atg5 in the Huayuqutan Recipe group was significantly increased(P<0.05);12h:There was no significant difference in the expression levels of Atg3 and Atg5 between the groups.Conclusion:1.Hep G2 cells could successfully replicate the lipid deposition model after being treated with a concentration of 1 mmol/L oleic acid and palmitic acid(molar ratio 2:1)for 24 h.2.Huayuqutan Recipe can effectively improve the level of lipid deposition induced by oleic acid and palmitic acid mixture in Hep G2 cells.3.Huayuqutan Recipe may effectively reduce the level of lipid deposition in Hep G2 cells induced by the mixture of oleic acid and palmitic acid by up-regulating the autophagy level of Hep G2 cells.Paper two:Huayuqutan Recipe can reduce the lipid deposition of Hep G2 cells induced by FFA on inhibiting the PI3K/AKT/m TOR signaling pathwayPurpose:Based on the PI3K/AKT/m TOR signal pathway,the possible mechanism of reducing the lipid deposition of Hep G2 cells induced by FFA induced by Huayuqutan Recipe is discussed.Material and method: Hep G2 cells cultured in vitro were randomly divided into 5groups: normal control group,model group,added to the culture medium at the concentrationof 1 mmol/L oleic acid and palmitic acid mixture(the molar ratio of 2 to 1),LY294002 treatment group,removing phlegm and Blood Stasis Decoction treatment group,removing phlegm and blood stasis decoction combined with LY294002 treatment group.Lipid deposition in Hep G2 cells was detected by oil red O staining and TG content;Immunofluorescence histochemical staining was used to observe the expression of microtubule associated protein 1 light chain 3(LC3)in each group;The levels of p-AKT,P-m TOR,LC3 and SREBP-1c in each group were detected by Western blot;The expression levels of SREBP-1c and LC3 m RMA were detected by Q-PCR.Results:1.Oil red O method show that compared with the normal control group,the lipid droplets in the model group showed a significant increase;Compared with the model group,the lipid droplets in the LY294002 group had no significant change,and the lipid droplets in the Huayuqutan Recipe group and the Huayuqutan Recipe group combined with the LY294002 group were significantly reduced.2.Photoelectric colorimetric method show that,compared with the normal control group,the content of TG cells in the model group increased significantly(P<0.05);compared with the model group,there was no significant change in TG content in the LY294002 group,and the TG content in the Huayuqutan Recipe group and Huayuqutan Recipe group combined with LY294002 group was significantly decreased(P< 0.05).3.Immunofluorescencemethod show that LC3 B was weakly expressed in the normal control group and LY294002 group,and was positively expressed in the model group.The strong positive expression of LC3 B was observed in the Huayuqutan Recipe group and Huayuqutan Recipe group combined with LY294002 group.4.Western Blot method show that compared with the normal control group,The protein levels of p-AKT and p-m TOR in the model group decreased significantly(P<0.05),and the expression of SREBP-1c protein and LC3 B protein increased significantly(P<0.05).Compared with the model group,p-AKT,p-m TOR,and LC3 B protein levels in the LY294002 group were significantly lower(P<0.05),and there was no significant change in SREBP-1c protein expression.The Huayuqutan Recipe group and Huayuqutan Recipe groupcombined with LY294002 The protein levels of p-AKT,p-m TOR,and SREBP-1c were significantly decreased(P<0.05),and the expression of LC3 B protein was significantly increased(P<0.05).5.Q-PCR showed that,compared with the normal control group,The expression of SREBP-1c and LC3 B m RNA in the model group was significantly increased(P<0.05).Compared with the model group,the expression of SREBP-1c m RNA in the LY294002 group was not significantly changed,and the expression of LC3 B m RNA was significantly decreased(P<0.05).The SREBP-1c m RNA was significantly increased in the Huayuqutan Recipe group and Huayuqutan Recipe group combined with LY294002 group.Decreased(P<0.05),LC3 B m RNA expression levels increased significantly(P<0.05).Conclusion:1.The mechanism of Huayuqutan Recipe can effectively improve the level of lipid deposition in Hep G2 cells induced by oleic acid and palmitic acid mixture,which may be related to inhibiting the expression of related proteins in PI3K/AKT/m TOR signaling pathway. |
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