Study On The Mechanism Of Berberine Ameliorate Insulin Resistance Via PPM1B Signaling Pathway

Objective To explore the mechanism of Berberine regulating PPM1 B signaling pathway to improve insulin resistance,and to provide evidence for targeted treatment for insulin resistance.Methods The proliferation of HepG2 cell which treated with different concentrations of insulin and different doses of berberine was detected by MTT method.HepG2-IR cell model was induced by high concentration of insulin and the chang of glucose consumption and glucose uptake were determined after Berberine(BBR)treatment by the glucose oxidase assay kit and fluorescent-glucose 2-NBDG.Concentration gradient of BBR(5,10,20 μg/ml)was added into insulin resistance HepG2 cells and the effect of berberine o n c AMP-PKA-PPM1 B pathway of concentration of c AMP in cells was detected by ELISA.The m RNA expression level of cellular inflammatory factor,anti-inflammatory factor and target gene of c AMP-PKA-PPM1B、PPM1B-GLUT4、PPM1B-IKKβ-NFκB、PPM1B-PI3K-AKT pathway determined by RT-PCR in HepG2-IR cells.Western blot is used to measure the target proteins expression level of c AMP-PKA-PPM1B、PPM1B-GLUT4、PPM1B-IKKβ-NFκB、PPM1B-PI3K-AKT pathway,the influence of NF-κB p65 protein nuclear translocation after treated with BBR and the influence for protein expression of PPM1 B with H-89.The protein expression of PPM1B、PPARγ、LRP1、GLUT4 、 IKKβ and NF-κB p65 also was detected by immunofluorescence in HepG2-IR cells.The HepG2 cells were infected with PPM1 B RNAi then establish insulin resistance model,and the unit cell glucose consumption was detected after being treated with BBR;The m RNA expression level of cellular inflammatory factor,anti-inflammatory factor and target gene of PPM1B-GLUT4、PPM1B-IKKβ-NFκB、PPM1B-PI3K-AKT pathway and the protein expression levels of PPM1B、PPAR-γ、GLUT4、p IKKβ Ser181、IKKβ、JNK and NF-κB p65 were also detected.Results Glucose consumption and uptake decreased in HepG2-IR cells by insulin-induced,and addition of berberine increases cellular glucose consumption and uptake(P<0.01).The result of ELISA showed that berberine can reduce the content of c AMP in insulin resistance cells,and the result of q-PCR showed that berberine can effectively inhibit the m RN A expressions of TNF-α,IL-1β,IL-6,IL-8 and promote the m RNA expressions of IL-10(P<0.01),which improved the inflammatory response in insulin resistance to some extent.The results of q-PCR showed that berberine promoted the m RN A expression of PPM1 B,PPARγ,LRP1,GLUT4,IRS-1,IRS-2,PI3 K,AKT,PDE3 B and PDE4 A yet the m RNA expression of JNK and NF-κB was inhibited(P<0.01).The results of Western blot showed that berberine promoted the protein expression of PPM1 B,PPARγ,LRP1,GLUT4,IRS-1,IRS-2,PI3 K p85and p AKT Ser473 and inhibit the expressio n of JNK,NF-κB p65,p IRS-1Ser 307,p IRS-2 Ser 731 and PKA(P<0.01 or P<0.05),but it had no difference on total protein expression of AKT and IKKβ(P>0.05).PKA inhibitor,H-89,effectively increased the expression of PPM1 B protein in HepG2 insulin resistance model(P<0.05);The results of immunofluorescence showed that PPM1 B,PPARγ,LRP1 and IKKβ mainly distributed in the cytoplasmic and nucleus,GLUT4 and NF-κB p65 mainly distributed in the cytoplasm and membrane of HepG2 cells.As the dose of berberine increased,the fluorescent expression of PPM1 B,PPARγ,LRP1,and GLUT4 increased,while the protein expression of NF-κB p65 decreased and berberine could effectively inhibit the nuclear translocation of NF-κB p65.In insulin resistance model HepG2 cells transfectd with PPM1 B lentivirus,the m RNA levels of PPM1 B and PPM1 A were significantly reduced,cell glucose consumption and uptake were decreased,the m RNA expression of inflammatory factors were increased(P<0.05),while the m RNA expression of PPM1 B,PPM1A,PPAR-γ,LRP1,GLUT4,IKKβ,IRS-1 and PI3 K were significantly decreased(P<0.01),and the protein expression of p IKKβSer181,IKKβ,JNK and NF-κB p65 were significantly increased(P<0.05).After the intervention with berberine(10μg/ml),the m RNA expression of TNF-α,IL-1β and IL-6 decreased significantly,yet the m RNA expression of anti-inflammatory factor,IL-10,was increased significantly(P<0.01).The glucose consumption of per unit cell and the ability of cells to absorb glucose increased significantly(P<0.01).The m RNA expression levels of PPM1B、PPM1A,PPAR-γ,LRP1,GLUT4,IKKβ,JNK,NF-κB,IRS-1 and PI3 K were significantly increased(P<0.01),while the protein expression of PPM1 B,PPARγ and GLUT4 improved significantly,while the protein e xpression of p IKKβ ser181,IKKβ,JNK and NF-κB p65 decreased significantly(P<0.05).Conclusion Berberine up-regulating significantly the m RN A and protein expression of PPM1 B in HepG2-IR cells.Berberine degrad ing c AMP to weaken the c AMP/PKA signal pathway and improving the expression of PPM1 B.Berberine inhibits the expression of inflammatory cytokines,reduce phosphorylation of IKKβand then inhibit activation of IKKβ possibly though PPM1 B to reduce the activation of inflammatory signaling pathway.Berberine promote s glucose consumption and uptake effectively may be improving insulin signal transduction pathway through PPM1 B.Therefore,the mechanism of berberine against insulin resistance may play an important role in resisting insulin resistance partly via PPM1 B,and PPM1 B may be one of the important targets to improve insulin resistance of HepG2 cells for berberine.