Effects Of Quercetin Regulated Plin1 Expression On Lipid Metabolism And AMPK/SREBP1 Pathway In NAFLD Mice

Objective: To observe the effects of different doses of quercetin on the expression of the Plin1 gene and lipid metabolism-related indicators in NAFLD mice model,and analyze the correlation between changes in Plin1 gene expression and lipid metabolism indicators.Additionally,we aim to utilize gene knockout technology to investigate the potential key role of the Plin1 gene in the process of quercetin intervention on lipid metabolism and the mRNA and protein expression changes in the AMPK/SREBP1 pathway.Methods: 1.To observe the effects of different doses of quercetin on the expression of the Plin1 gene and lipid metabolism-related indicators in NAFLD mice model,and analyze the correlation between changes in Plin1 gene expression and lipid metabolism indicators,NAFLD model was established by feeding mice 60% high-fat diet.The control group consisted of 8 mice fed standard maintenance diet,while NAFLD model group consisted of 8 mice fed 60% high-fat diet.After 12 weeks of feeding,the mice were euthanized using cervical dislocation after blood collection from the orbital sinus.Serum levels of TC,TG,ALT,AST,as well as liver tissue TC and TG,were measured.Liver tissue morphology was observed,and semi-quantitative analysis of liver pathology slides was conducted to calculate the NAS score.A comprehensive comparison of the aforementioned indicators between NAFLD model group and the control group was performed to confirm the successful construction of the model.The experimental animals were divided into 6 groups using random number table method,comprising 48 SPF-grade male C57BL/6J mice aged 4 weeks.The groups included: control group(C group),consisting of 8 mice fed standard maintenance diet with daily administration of physiological saline by gavage;NAFLD model group(NAFLD group),consisting of 8 mice fed a 60% high-fat diet with daily administration of physiological saline by gavage(the 0-dose intervention group);and different doses of quercetin intervention groups,each consisting of 8 mice fed 60% high-fat diet.The quercetin intervention groups were divided based on the intervention dose: 50 mg/(kg·bw)/d dose group(50mg/kg group),receiving daily gavage of 5 mg/m L quercetin solution(gavage solution volume calculated based on dosage of 10 μl/g of mice body weight);100 mg/(kg·bw)/d dose group(100mg/kg group),receiving daily gavage of 10 mg/m L quercetin solution(gavage solution volume calculated as above);200 mg/(kg·bw)/d dose group(200mg/kg group),receiving daily gavage of 20 mg/m L quercetin solution(gavage solution volume calculated as above);and 400 mg/(kg·bw)/d dose group(400mg/kg group),receiving daily gavage of 40 mg/m L quercetin solution(gavage solution volume calculated as above).The mice were weighed weekly,their food intake was measured daily,and their growth,mental state,and survival were observed daily.After 12 weeks of feeding,the mice were fasted overnight for 12 hours,then euthanized using cervical dislocation,real-time fluorescence quantitative PCR and immunohistochemistry were used to assess changes in the expression of Plin1 mRNA and protein in liver tissue.The enzyme-linked immunosorbent assay was performed according to the instructions of the kit to measure serum levels of TC,TG,ALT,AST,as well as liver tissue TC and TG.Hepatic morphology was observed using hematoxylin and eosin(HE)staining,and semi-quantitative analysis of liver pathology slides was conducted to calculate the NAS score.Oil Red O staining was used to observe the distribution of lipid droplets in the liver.SPSS Pearson correlation analysis was employed to analyze the correlation between changes in Plin1 gene expression and lipid metabolism indicators.Based on the results of the measurements,the optimal dosage of quercetin were selected for the next experiment.2.The effects of Plin1 gene knockout on quercetin intervention in NAFLD mice model were observed.Homozygous Plin1 gene knockout mice,identified through tail DNA analysis,were selected from the purified strain of knockout mice as experimental subjects.The experimental animals were divided into three groups: the control group(C group)consisted of 8 male mice fed a regular maintenance diet and administered physiological saline by gavage daily;the NAFLD model group(NAFLD group)consisted of 8 male mice fed 60% high-fat diet and administered physiological saline by gavage daily;400 mg/(kg · bw)/d quercetin intervention group(400mg/kg group)consisted of 8 male mice fed 60% high-fat diet and administered 40 mg/m L quercetin solution by gavage daily(with gavage solution volume calculated as before).In addition,24 male SPF-grade C57BL/6J mice aged 4 weeks,with Plin1 gene knockout,were adapted to the environment for one week and randomly divided into three groups: the knockout control group(KO-C group)consisted of 8 knockout mice fed regular maintenance diet and administered physiological saline by gavage daily;the knockout model group(KO-N group)consisted of 8 knockout mice fed 60% high-fat diet and administered physiological saline by gavage daily;the 400 mg/(kg·bw)/d quercetin intervention group with Plin1 gene knockout(KO-400mg/kg group)consisted of 8 knockout mice fed 60% high-fat diet and administered 40 mg/m L quercetin solution by gavage daily.The mice were weighed weekly,their food intake was measured daily,and their growth,mental state,and survival were observed daily.After 12 weeks of feeding,the mice were fasted overnight(with access to water)for 12 hours and then euthanized using cervical dislocation.The contents of TC,TG,AST and ALT in serum and liver were measured.The liver weight was measured to calculate the liver index.Hepatic morphology was observed using HE staining,and semi-quantitative analysis of liver pathology slides was conducted to calculate the NAS score.Oil Red O staining was used to observe the distribution of lipid droplets in the liver.3.The effects of Plin1 gene knockout on the expression of AMPK/SREBP1 pathway-related mRNA and protein in the liver tissue of NAFLD model mice under quercetin intervention,the gene knockout mice model construction,identification,and animal grouping were performed as described in the second part.Real-time fluorescence quantitative PCR and immunohistochemistry were used to detect the changes in AMPK,SREBP1,FASN,SCD1,ACC1 mRNA and protein expression in the AMPK/SREBP1 pathway,the specific detection methods were the same as described in the first part.Results: 1.During the feeding and gavage period,all groups of mice exhibited good mental status,and no deaths occurred.Compared to C group,the NAFLD group showed significant decrease in weekly food intake(P<0.05),significant increase in body weight(P<0.05),significant elevation in serum TC,TG,ALT,AST,and liver TC,TG levels(P<0.05),apparent enlargement of the liver with a pale color,rough surface,and greasy texture,significant decrease in the liver index(P<0.05),evident presence of lipid vacuoles and fat degeneration cells in the liver,as well as inflammatory infiltration,nuclear compression and deformation towards the cell membrane,semi-quantitative analysis indicating NAS score >4,and oil red O staining revealing significant increase and dense distribution of lipid droplets(P<0.05).Taken together,these results indicate the successful construction of the NAFLD model.The results of real-time fluorescence quantitative PCR and immunohistochemistry showed significant increase in Plin1 mRNA and Plin1 protein expression in NAFLD group compared to the other groups(P<0.05).In comparison to NAFLD group,quercetin intervention led to decrease in body weight in the quercetin intervention groups.The expression of Plin1 mRNA and Plin1 protein in the quercetin intervention groups showed dose-dependent decrease.Serum TC,TG,ALT,AST,and liver TC,TG levels decreased successively,with the most significant effects observed in 400mg/kg group(P<0.05).In the quercetin intervention groups,there was marked reversal of hepatocellular steatosis compared to NAFLD group,with reduction in lipid vacuoles and significant decrease in lipid droplets,especially in 400mg/kg group(P<0.05).Statistical correlation analysis revealed significant positive correlations between changes in serum TC,TG,ALT,AST,liver TC,TG levels,and the expression trends of Plin1 mRNA and protein.Among them,the correlations between changes in serum TC,TG,AST,ALT,liver TC,TG levels,and Plin1 protein expression were more prominent,with r-values closer to 1.2.During the experiment,no deaths occurred in any of the groups,and the mice exhibited good mental status,adequate food intake,and agile motor responses.The mice had smooth and glossy fur.The results of the lipid metabolism level analysis showed the following: Compared to C group,NAFLD group exhibited significant increase in serum and liver tissue levels of TC,TG,ALT,AST(P<0.05).The liver appeared enlarged with darker red color,firmer texture,and significant increase in liver weight(P<0.05).Fat vacuoles and fat degeneration cells were significantly present in the field of view.The NAS score significantly increased(P<0.05),and oil red O staining revealed increase in lipid droplets(P<0.05).Compared to NAFLD group,the 400mg/kg group showed significant decrease in serum and liver tissue levels of TC,TG,ALT,AST(P<0.05),liver weight significantly decreased(P<0.05),the phenomena of fat vacuoles and fat degeneration cells were effectively reversed,and inflammatory infiltration reduced,the NAS score significantly decreased(P<0.05),and oil red O staining showed significant reduction in lipid droplets(P<0.05).Compared to the 400mg/kg group,the KO-400mg/kg group exhibited significant increase in serum and liver tissue levels of TC,TG,ALT,AST(P<0.05),and some indicators even had absolute values higher than the NAFLD group,mice liver weight significantly increased(P<0.05),and liver index significantly increased(P<0.05).In terms of tissue morphology,there was no alleviation of hepatic steatosis observed in the liver tissue,and there were still evident fat vacuoles and fat degeneration cells,the NAS score significantly increased(P<0.05),and oil red O staining revealed significant increase in lipid droplets(P<0.05).In addition,compared to C group,KO-C group showed significant increase in serum and liver tissue levels of TC,TG,ALT,AST(P<0.05),although the degree of increase was mostly lower than that of NAFLD group.When knockout and high-fat modeling were combined(KO-N group),TC,TG,ALT,AST levels significantly increased(P<0.05),and the increase was higher than that in NAFLD group.When quercetin intervention was administered to the KO-N group(KO-400mg/kg group),there was no significant decrease in TC,TG,ALT,AST levels,indicating that the differences in TC,TG,ALT,AST changes between the KO-400mg/kg group and the KO-N group were not significant.3.The results of the detection of AMPK/SREBP1 pathway-related mRNA and protein expression levels in each group of mice are as follows: Compared to C group,KO-C group and NAFLD group showed significant decrease in AMPK mRNA and phosphorylated protein expression(P<0.05),while SREBP1,FASN,SCD1,and ACC1 mRNA and protein expression showed significant increase(P<0.05).After intervention with quercetin,400mg/kg group showed significant increase in AMPK mRNA expression and phosphorylated protein(P<0.05).while SREBP1,FASN,SCD1,and ACC1 mRNA and protein expression showed significant decrease(P<0.05).Compared to 400mg/kg group,KO-400mg/kg group showed significant decrease in AMPK mRNA and phosphorylated protein(P<0.05),while SREBP1,FASN,SCD1,and ACC1 mRNA expression showed significant increase(P<0.05).In the comparison between knockout groups,the KO-N group showed significant decrease in AMPK mRNA and phosphorylated protein expression compared to the KO-C group(P<0.05),while SREBP1,FASN,SCD1,and ACC1 mRNA and protein expression showed significant increase(P<0.05).Compared to NAFLD group,KO-N group showed further decrease in AMPK mRNA and phosphorylated protein expression(P<0.05),while SREBP1,FASN,SCD1,and ACC1 mRNA and protein expression showed further increase(P<0.05).After intervention with quercetin,KO-400mg/kg group showed increase in AMPK mRNA and protein expression,but the difference was not significant.Although there was certain decrease in SREBP1,FASN,SCD1,and ACC1 mRNA and protein expression,the difference was also not significant.Conclusion: Quercetin intervention can effectively alleviate a series of negative changes in the NAFLD model mice,knocking out the Plin1 gene eliminates the protective effects of quercetin intervention,which may affect lipid metabolism and AMPK/SREBP1 pathway in NAFLD model mice by regulating Plin1 gene expression.