| Objectives:1. To observe the effect on the activity of H9C2 cells induced by high glucose.2. To observe the effect of curcumin on the activity and inflammation of H9C2 ells induced by high glucose.3. To explore the protective effects of curcumin on the inflammation of H9C2 ells induced by high glucose and its mechanisms.Methods:1. The H9C2 cells were cultured in vitro with low sugar medium, and then to ntervene with high sugar, curcumin, liver X agonists/inhibitors, TLR inhibitors, PP2A nhibitors respectively.2. The experimental groups divided as follows:①Control groups:The H9C2 cells were cultured in low sugar culture medium(5.5mmol/L);②High glucose group:The H9C2 ells were cultured in high sugar culture medium(33mmol/L);③ igh glucose+T0901317 group:Intervented with liver X agonists(T0901317 final concentration was 20[^mol/L) on he basis of high glucose group;④ High glucose+TAK-242 group:Intervented with TLR nhibitors(TAK-242 final concentration was 100nmol/L) on the basis of high glucose group;⑤High glucose+Curcumin group:Intervented with curcumin(Curcumin final concentration was 10μmol/L) on the basis of high glucose group;⑥igh glucose+ 5CPPSS-50 group:Intervented with liver X inhibitor(5CPPSS-50 final concentration was I5μmol/L) on the basis of high glucose group;⑦High glucose+5CPPSS-50+Curcumin group:Intervented with Curcumin on the basis of high glucose+5CPPSS-50 group;⑧igh glucose+Okadaic acid group:Intervented with PP2A inhibitor(Okadaic acid final concentration was 20nmol/L) on the basis of high glucose group;⑨High glucose+ Okadaic acid+Curcumin group:Intervented with Curcumin on the basis of high glucose+ )kadaic acid group.3.72 hours after the appropriate drug intervention. The rate of H9C2 cells viability of each group was determined by CCK-8 detection kit.Detected the inflammatory cytokines levels of IL6,TNFa and MCP1 by enzyme-linked mmunosorbent assay. Genes expression of LXRa,IRAK4 and PP2Ac detected by qRT-PCR. Western blot was used to discover the expression levels of NF-κB p65 (whole ell lysate and nuclear protein), LXRa, IRAK4 and p-PP2Ac.Results:1. The cells viability of each H9C2 cardiomyocyte groups was detected by CCK-8,the results showed that:High glucose can inhibit the proliferation activity of H9C2 cardiomyocyte. The inhibition of proliferation activity weaked by LXRs igonists, TLR inhibitors or curcumin. The inhibition of proliferation activity ntensified after LXRs inhibitors or PP2A inhibitors intervention. Based on intervented with LXRs inhibitors or PP2A inhibitors, the use of curcumin could not improve the nhibition of proliferation activity of H9C2 cell induced by high sugar.2. The secretion of inflammatory cytokines(IL6, TNFa and MCP1)in the supernatant measured by enzyme-linked immunosorbent assay showed that:The nflammatory cytokines levels of high glucose cell culture supernatant were significantly increased (P<0.05). The secretion of inflammatory cytokines reduced after T0901317, TAK-242 or Curcumin intervented (P<0.05).5CPPSS-50 or Okadaic cid exacerbated inflammatory cytokine secretion (P<0.05). Compared with high glucose+curcumin group, based on intervented with LXRs inhibitors or PP2A nhibitors, the use of curcumin could not reduce inflammatory factors secretion P<0.05).3. The mRNA expression of LXRa, IRAK4, PP2Ac were detected by qRT-PCR, he results showed that:High glucose can increase the mRNA expression level of LXRa slightly, but there was no statistically difference(P>0.05), meanwhile the nRNA expression levels of IRAK4 and PP2Ac were significantly increased (P<0.01) Compared with HG group:the expression of LXRa mRNA further increased after the ntervention of T0901317,TAK-242 or Curcumin(P<0.01), but the expression of RAK4 mRNA decreased significantly(P<0.01);HG+5CPPSS-50 group result suggested that the mRNA expression of LXRa inhibited significantly(P<0.01), nstead, the mRNA expression of IRAK4 increased(P<0.01). On the basis of intervented with LXRs inhibitors, the use of curcumin could not upregulation the expression of LXRa mRNA. In addition, compared with the HG group also showed: he mRNA expression of PP2Ac was further increased significantly after PP2A nhibitors intervented(P<0.01), curcumin can reduce the mRNA expression of PP2Ac significantly (P<0.01), but on the basis of intervented with PP2A inhibitors, the unction of curcumin downregulated the mRNA expression of PP2Ac weakened significantly.4. The protein expression of NF-κB p65(whole cell lysate and nuclear protein), LXRα, IRAK4 and p-PP2Ac were detected by western blot, the results showed that: The protein level of NF-κB p65 in total cell lysate had no statistically difference in each group(P>0.05). High glucose can increase the protein expression level of LXRα slightly, but there was no statistically difference(P>0.05), meanwhile the protein expression levels of NF-κB p65(nuclear), IRAK4 and p-PP2Ac increased significantly(P<0.01). Compared with HG group:the protein expression of LXRα is urther increased after the intervention of T0901317,TAK-242 or Curcumin(P<0.05), put the expression of IRAK4 protein and NF-κB p65 nuclear protein were decreased significantly(P<0.05); HG+5CPPSS-50 group result suggested that the protein expression of LXRα was inhibited(,P<0.05), instead, the expression of IRAK4 protein and NF-κB p65 nuclear protein increased significantly(P<0.01). On the basis of ntervented with LXRs inhibitors, the use of curcumin could not upregulation the expression of LXRa protein and downregulation NF-κB p65 nuclear protein. In iddition, compared with the HG group also showed:the expression of p-PP2Ac rotein and NF-κB p65 nuclear protein further increased significantly after PP2A nhibitors intervented(P<0.05), curcumin can reduce the expression of p-PP2Ac rotein significantly(P<0.01), but on the basis of intervented with PP2A inhibitors, the unction of curcumin downregulated the expression of p-PP2Ac protein and NF-κB p65 nuclear protein weakened significantly.Conclusions:1. High glucose can induce H9C2 cardiac cells inflammation reaction, curcumin can inhibit high glucose-induced inflammation reaction.2. Curcumin can increase the expression of LXRa and inhibit the expression of RAK4 and p-PP2Ac, activating the function of PP2A negatively regulate TLR-NF-κB pathway, followed by NF-κB p65 translocation into the nucleus lecreased, resulting in inhibited transcription of responsive inflammatory cytokines IL6, TNFa and MCP1), which may result in the high glucose-induced H9C2 cardiomyocytes inflammation alleviation by this effect.It suggests that the integration of LXRs,TLR-NF-κB pathway, associated protein kinase and phosphatases may be he cellular and molecular mechanism of curcumin reduce the high glucose-induced H9C2 cardiomyocytes inflammatory response. |
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