A Study Of Isoquercitrin To Ameliorate Insulin Resistance By Circular HIPK3 Inhibiting MiR-29a-3p Via Regulating PI3K/AKT Signaling Pathway

Objective:To investigate the mechanism of isoquercitrin inhibiting miR-29a-3p through circ HIPK3 and activating the PI3K/AKT signaling pathway to improve insulin resistance.Methods:1.In vitro experiments:（1）The m RNA expression levels of circ HIPK3 and HIPK3 in Hep G2 cells after RNase R treatment were detected by q RT-PCR assay.（2）1×10^-7mol/L insulin and 125μM palmitic acid were used to construct a Hep G2 cell insulin resistance model,The expression of circ HIPK3 and miR-29a-3p with pinsulin resistance in Hep G2 cells by Quantitative Real Time-PCR（q RT-PCR）method;（3）Using bioinformatics analysis method to predict whether circ HIPK3 has a targeting relationship with miR-29a-3p,and verify whether circ HIPK3 can target miR by dual-luciferase method（Luciferase）-29a-3p activity;（4）After transiently transfecting circ HIPK3to treating insulin-resistant Hep G2 cells with isoquercitrin of 48 hours later that the isoquercein effect of cortex glycosides on the m RNA and protein expression of target genes in insulin-resistant Hep G2 cells overexpressing circ HIPK3 by Western Blot（WB）,glucose oxidase method and q RT-PCR;（5）Co-transfection of circ HIPK3 and miR-29a-3p simultaneously treated insulin-resistant Hep G2 cells with isoquercitrin and transfected after 48 h of staining,it is used to detect the inhibitory effect of isoquercitrin on miR-29a-3p through circ HIPK3,activate the expression of each target gene in the PI3K/AKT signaling pathway by WB method,glucose oxidase method and q RT-PCR,and improve cellular insulin resistance.2.In vivo experiment:（1）Take 18 SPF db/db mice and divide them into normal control group,diabetes group,empty plasmid adenovirus group,circ HIPK3overexpression adenovirus group and another normal control group.The empty plasmid adenovirus and circ HIPK3 overexpressing adenovirus were injected into the tail vein of the empty plasmid adenovirus group and the circ HIPK3overexpressing adenovirus group respectively.The injection concentration was1E+10PFU/m L.After 14 days,In mouse liver tissue,the expression of circ HIPK3was detected by q RT-PCR experiment.（2）Another 36 SPF db/db mice were randomly divided into 6 groups（diabetes group,empty plasmid adenovirus control group,model group,low,medium and high doses of isoquercitrin）,and a normal control Group,the empty plasmid adenovirus control group,tail vein injection of empty plasmid adenovirus,the diabetic group and the normal control group,the tail vein injection of 0.9%normal saline,and the other 4 groups,the tail vein injection of circ HIPK3 overexpressing adenovirus.After 14 days,they were given intragastric administration.Different doses of isoquercitrin were respectively（20mg/kg,40 mg/kg,80 mg/kg）,and the rest were given an equal volume of 0.9%normal saline for 14 days.day.The fasting blood glucose of mice was monitored on day 0,day 7 and day 14 of administration.After the 14th day of intragastric administration,samples were taken,and the serum was taken for enzyme-linked immunosorbent assay（ELISA）to measure the fasting insulin content of mice.Take the pancreas tissue to make paraffin sections for H-E and immunohistochemical staining experiments.The liver tissue was taken to extract RNA and protein,and the expression of various factors in the miR-29a-3p and PI3K/AKT pathways in mice were detected by WB technology and q RT-PCR technology.Results:1.In vitro experiment:（1）The expression level of circ HIPK3did not significantly change before and after RNase R treatment,but the expression level of linear m HIPK3 was significantly decreased,which confirmed that circhip K3 had a circular structure and stable structure.（2）Compared with the control group,the expression of circ HIPK3 in the model group was significantly reduced,and the expression of miR-29a-3p was significantly increased,and the difference was statistically significant.（3）The dual luciferase experiment indicated that the fluorescence intensity of the wild-type plasmid group of circ HIPK3 was significantly increased,while the fluorescence intensity of the mutant plasmid group had no obvious change.circ HIPK3 has a targeting relationship with miR-29a-3p.（4）In the experiment of insulin resistant Hep G2cells transfected with circ HIPK3 plasmid,the expression level of miR-29a-3p in the group transfected with circ HIPK3 plasmid was lower than that in the isoquercitrin treatment group;PIK3R1,AKT and GLUT4 m RNA and protein expression levels Significant increase,while the m RNA and protein expression levels of FOXO1,GSK3βand PTEN decreased significantly,and the difference was statistically significant.（5）Co-transfection of circ HIPK3 and miR-29a-3p mimic into isoquercitrin-pretreated insulin resistant Hep G2 cells,compared with the isoquercitrin-treated group,co-transfection of circ HIPK3 and miR-29a-3p mimic The expression level of miR-29a-3p decreased;the m RNA and protein expression levels of PIK3R1,AKT and GLUT4 were significantly increased,while the m RNA and protein expression levels of FOXO1,GSK3βand PTEN were significantly decreased.The results of the grape oxidase method showed that compared with the isoquercitrin treatment group,the glucose consumption of co-transfected circ HIPK3 plasmid and miR-29a-3p mimic was significantly increased.2.In vivo experiments:（1）After the tail vein injection of circ HIPK3overexpressing adenovirus in db/db mice,the results of q RT-PCR showed that the expression of circ HIPK3 in the liver tissue of mice was higher than that in the diabetic group.（2）After 14 days of intragastric administration of different doses of isoquercitrin,compared with the circ HIPK3 overexpression adenovirus model group,the fasting blood glucose of mice in the different doses of isoquercitrin group decreased.The results of the ELISA experiment indicated that compared with the circ HIPK3 overexpression adenovirus model group,the fasting insulin content of mice in the different doses of isoquercitrin group increased.HE staining results showed that the morphological contours of pancreatic islets in the different doses of isoquercitrin group were relatively clear and regular,the size of pancreatic islet nuclei was more uniform,and the arrangement was more regular;the results of immunohistochemical staining showed that compared with the circ HIPK3 overexpression adenovirus model group,The expression of PIK3R1factor increased in different doses of isoquercitrin group,which activated the PI3K/AKT pathway.（3）The results of q RT-PCR and WB experiments showed that compared with the circ HIPK3 overexpression adenovirus model group,the expression of miR-29a-3p in the isoquercitrin group was decreased;in the different dose groups of isoquercitrin The m RNA and protein levels of PIK3R1,AKT,and GLUT4 increased significantly,while the m RNA and protein levels of FOXO1,GSK3βand PTEN decreased significantly.Conclusions:Isoquercitrin can regulate PI3K/AKT signaling pathway by acting on circ HIPK3 to inhibit miR-29a-3p and improve insulin resistance.