Effect And Mechanism Of Gegen Qinlian Decoction Alleviating Hepatic Endoplasmic Reticulum Stress To Improve Glucose Metabolism In Vitro And In Vivo

Objective:To investigate the effect and mechanism of Gegen Qinlian Decoction(GQD)alleviating hepatic endoplasmic reticulum stress(ERS)to improve glucose metabolism in vitro and in vivo.This study would be helpful to deeply understand the hypoglycemic effect and mechanism of GQD to provide theoretical basis and scientific basis for clinical treatment of type 2 diabetes(T2DM).Methods:1.Molecular docking predicted the binding ability of important GQD compounds and ERS-relative proteinsGQD compounds were searched using the Pub Chem(https://pubchem.ncbi.nlm.nih.gov/)then,were saved as SDF format.The compound SDF format was converted to PDB format using Open Babel 2.3.2.The crystal structure of ERS-relative proteins were downloaded from the PDB(https://www.rcsb.org/)database,with the assistant of Uniprot(https://www.uniprot.org/)database to define proteins as follow: GRP78(6DWS),IRE1(7BMK),PERK(4X7K),and ATF4(1CI6).The results were imported into Autodock 4.2.6,removing the water molecules and side chain residues,the main components including baicalin,berberine,daidzein,jatrorrhizine,liquiritin,palmatine,puerarin and wogonoside of GQD were imported.The docking scores of target and active ingredients were obtained by empirical scoring function.The score lower than-4.25 indicated that the active ingredient had a certain binding ability.The score which is lower than-5.0 indicates a good binding ability.2.Molecular mechanism of GQD relieving ERS in liver tissue of diabetic Sprague-Dawley(SD)ratsThe frozen liver tissue samples of diabetic rats were obtained in the previous experiment of our research group including the normal,model,positive and GQD administration group,the protein and m RNA were extracted respectively for further experiments.(1)q PCR was used to detect the m RNA expression of Grp78,Ire1 and Atf6 in liver tissue of diabetic rats treated with GQD.(2)q PCR was used to detect the m RNA expression of apoptosis-related genes Atf4,Chop and Caspase12 in liver tissue of diabetic rats treated with GQD.(3)Western blot(WB)was used to detect the protein expression of ERSrelated proteins GRP78,IRE1,PERK,ATF6 and XBP1 s in liver of diabetic rats treated with GQD.(4)WB was used to detect the protein expression of apoptosis-related proteins ATF4,CHOP,Caspase12,Caspase9 and Caspase3 in liver of diabetic rats treated with GQD.(5)Gene Symbol,Fold Change and P-value data which were included in the excel table was imported into IPA(Ingenuity Pathway Analysis)software for pathway analysis.The core analysis module was used to analyze and output the correlation results.(6)The liver tissues were used to detect calcium content according with the instructions of calcium ion detection kit.3.Effect and Mechanism of GQD Containing Serum(GQD-CS)in relieving ERS to improve IR in tunicamycin-induced IR-Hep G2 cells in vitro.Hep G2 cells were induced with 2 μM Tunicamycin(Tm)alone for 6 h to establish a stable IR-Hep G2 cell model.The IR-Hep G2 cells were divided into control group,IR model group,positive group(2 m M metformin)and GQD-CS administration group(2.5%,5%,10%)with 9 h treatment.(1)The extracellular glucose of IR-Hep G2 cells were detected by glucose oxidase(GOD-POD).(2)The glycogen of IR-Hep G2 cells were detected by PAS staining.(3)The cell viability of IR-Hep G2 cells were detected by CCK-8 assay.(4)The apoptosis rate of of IR-Hep G2 cells were detected by flow cytometry.(5)ERS marker GRP78 was detected by immunofluorescence(IF).(6)The protein expression of ERS-related GRP78,IRE1,ATF6,PERK and p-e IF2α(Ser51)were detected by WB.(7)The protein expression of insulin hypoglycemic pathway related proteins p-JNKs(Thr183/Tyr185),p-IRS1(Ser312),p-PI3Kp85(Tyr607)and p-Akt(Ser473)were detected by WB.(8)The calcium content was detected strictly according to the instructions.Results:1.The docking results of GQD active constituents with the important ERS proteins target molecules of ERS.(1)GRP78(6DWS)showed good binding activity with baicalein(-8.4),berberine(-7.1),daidzein(-7.5),jatrorrhizine(-7.9),liquiritin(-8.9),palmitine(-7.4),puerarin(-9.1)and wogonoside(-9.3).(2)IRE1(7BMK)showed good binding activity with baicalein(-9.6),berberine(-8.6),daidzein(-8.5),jatrorrhizine(-9.4),liquiritin(-9.6),palmitine(-8.7),puerarin(-9.6),and wogonoside(-9.5).(3)PERK(4X7K)showed good binding activity with baicalein(-7.5),berberine(-7.3),daidzein(-7.0),jatrorrhizine(-7.0),liquiritin(-8.3),palmitine(-7.4),puerarin(-7.5)and wogonoside(-7.7).(4)ATF4(1CI6)showed good binding activity with baicalein(-6.9),berberine(-6.7),daidzein(-6.0),jatrorrhizine(-6.9),liquiritin(-6.8),palmitine(-6.5),puerarin(-7.0),and wogonoside(-7.2).2.Molecular mechanism of GQD alleviated ERS in liver tissue of diabetic rats.(1)Grp78,Ire1 and Atf6 were up-regulated in diabetic model group(P<0.001).Moreover,GQD down-regulated the m RNA expression of Grp78(P<0.001),Ire1 and Atf6(P<0.05).(2)Atf4(P<0.05),Chop and Caspase12(P<0.001)were up-regulated in diabetic model group.Moreover,GQD down-regulated the m RNA expression of Atf4(P<0.05),Chop and Caspase12(P<0.001).(3)GRP78,IRE1 and PERK were up-regulated in the diabetic model group(P<0.001),Moreover,GQD down-regulated the protein expression of GRP78,IRE1 and PERK(P<0.001),and ATF6 had an increasing trend,which could promote the adaptive UPR with the up-regulation of XBP1 together(P<0.05).These results suggest that GQD alleviated ERS-induced UPR in liver of diabetic rats.(4)ATF4 、 CHOP,Caspase12,Caspase9 and Caspase3 were up-regulated.Moreover,GQD down-regulated the protein expression of ATF4,CHOP,Caspase12,Caspase9 and Caspase3(P<0.001).These results suggested that GQD inhibited hepatocyte apoptosis induced by ERS in liver tissue of diabetic rats.(5)IPA classical pathway analysis of protein expression results mainly involved in ERS-UPR pathway.IPA disease and function analysis showed that the differentially expressed genes were associated with abnormal protein folding,suggesting that GQD alleviated hepatic ERS to promote protein folding.(6)The calcium content in liver tissue of diabetic rats was down-regulated whereas GQD up-regulated the content of calcium to alleviate the imbalance of calcium homeostasis.3.The hypoglycemic effect and mechanism of GQD-CS alleviating excessive ERS to improve hepatic insulin sensitivity in tunicamycin-induced IR-Hep G2 cells.(1)GQD-CS promoted the hypoglycemic effect of tunicamycin-induced IRHep G2 cells induced with ERS:2.5%,5% and 10% GQD-CS down-regulated the extracellular glucose content(P<0.05).(2)PAS staining results showed that GQD-CS up-regulated the glycogen content.(3)GQD-CS did not affect tunicamycin-induced IR-Hep G2 cell viability for 9h with GQD-CS treatment by CCK-8 assay.(4)GQD-CS effectively down-regulated the apoptosis rate of tunicamycininduced IR-Hep G2 cells by flow cytometry.(5)2.5%,5% and 10% GQD-CS down-regulated GRP78 expression level in tunicamycin-induced IR-Hep G2 cells by IF.(6)GQD-CS down-regulated GRP78 and PERK(P<0.01),IRE1、ATF6 and p-e If2α(Ser51)(P<0.05)in tunicamycin-induced IR-Hep G2 cells.(7)GQD-CS activated p-JNKs(Thr183/Tyr185)、p-PI3KP85(Tyr607)and pAkt(Ser473)(P<0.05),and inhibited p-IRS1(Ser312)(P<0.05)in tunicamycininduced IR-Hep G2 cells to improve glucose metabolism.(8)GQD-CS effectively up-regulated the calcium content in tunicamycininduced IR-Hep G2 cells.Conclusions:GQD alleviated hepatic UPR and cell apoptosis induced by excessive ERS,increased calcium content,which contributed to the homeostasis of endoplasmic reticulum function,thereby GQD improved hepatic insulin signaling pathway to regulate glucose metabolism in vivo and in vitro.