| Objective To investigate the role and mechanism of HDAC3 in the regulation of immune response and insulin secretion by Lycium Barbarum polysaccharide(LBP).Methods(1)SD rats were divided into control group,diabetes mellitus(DM)group,LBP low(20mg/kg),medium(40mg/kg),high(80mg/kg)intervention group(n=8 in each group).The relationship between IRI,serum inflammatory and anti-inflammatory factors and HDAC3 gene in T2 DM rats was analyzed by detecting the blood glucose and INS levels,serum inflammatory factors,TNF-a,anti-inflammatory factors IL-10 and IL-4 levels,and the expression of HDAC3 gene in liver cells.(2)RAW264.7 macrophages were treated with curcumin(CUR)which was an inhibitor of HDAC3.LPS was used to induce inflammation in all groups.Blank control group was set up,as well as cur(8μg/m L),LBP intervention group(25,50,100μg/m L),LBP(25,50,100μg/m L)+ cur(8μg/m L).The expression of TLR4,NF-κB and p-NF-κB genes and protein was detected.The differences of TLR4,NF-κB and p-NF-κB expression before and after HDAC3 inhibition were compared,and the mechanism of HDAC3 regulating the immune response was analyzed.(3)After the inhibition of HDAC3 activity by curcumin(CUR),LBP intervened β-TC6 cells for 24 hours.Blank control group was set up,as well as HDAC3 inhibitor model group,LBP intervention group(25,50,100μg/m L),LBP(25,50,100μg/m L)and CUR(8μg/m L)group.The expression of INS,Islet-1 and Pdx-1 was detected by real time PCR.The Western blot method was used to detect the expression of INS,Islet-1 and PDX-1 proteinss before and after HDAC3 was inhibited,and the mechanism of HDAC3 regulating insulin secretion was analyzed.Results 1.Role and mechanism of HDAC3 in Lycium barbarum polysaccharide regulating immune response to type 2 diabetes mellitus(1)The liver index and TNF-α levels of diabetic rats were significantly higher than those of control group and LBP intervention group(P(27)0.05),while the serum IL-4 and IL-10 levels were lower than the control group and LBP intervention group.(2)Compared with the normal control group,LBP significantly decreased the transcription level of HDAC3 m RNA in the liver of T2 DM rats(P(27)0.01).Spearman correlation analysis showed that the transcription level of HDAC3 m RNA in liver was correlated with serum IL-4 level in T2 DM rats(R= 0.4295,P= 0.015).In addition,there was a correlation between LBP dose and HDAC3 m RNA transcription(R=0.8883,P=0.0001).Our study showed that LBP improved the inflammation of diabetes,and HDAC3 activity also changes.(3)RAW264.7 cells were induced by LPS.The results showed that LBP and curcumin(CUR)inhibited HDAC3 activity in inflammatory environment.(4)LBP and CUR inhibited the protein expression of NF-κB,p-NF-κB and TLR4 under inflammatory conditions(P(27)0.05).Cur combined with LBP also inhibited the protein expression of NF-κB and p-NF-κB(P(27)0.05).It was suggested that LBP can reduce the expression of inflammatory factors in macrophages induced by LPS.(5)Chip experiment showed that LBP and cur could reduce the level of H3 acetylation of TLR4 promoter in RAW264.7 macrophages induced by LPS(P(27)0.05).2.Effect and mechanism of HDAC3 on insulin secretion in type 2 diabetic rats induced by LBP.(1)After 24 hours of LBP intervention,IRI and liver index in LBP intervention group were significantly lower than those in model group(P(27)0.05).The results showed that LBP could reduce insulin resistance in T2 DM rats.(2)The expression of HDAC3 activity in liver nuclei of type 2 diabetic rats was detected.Compared with the control group and T2 DM group,LBP significantly promoted the expression of HDAC3 in liver nuclear protein of T2 DM rats(P(27)0.05).Compared with T2 DM group,LBP significantly decreased the activity of acetylated H3 in liver nuclei of T2 DM rats(P(27)0.01).Spearman correlation analysis showed that HDAC3 was correlated with IRI(R= 0.5392,P= 0.0368).There was no correlation between HDAC3 and liver index or histone acetylase 3.Our study shows that LBP can improve insulin resistance in diabetes,but also change the activity of HDAC3.(3)The activity of HDAC3 in β-TC cells was detected with or without glucose intervention.In the absence of glucose intervention,cur inhibited HDAC3 activity,while LBP50,LBP100 and LBP25 combined with cur enhanced HDAC3 activity(P(27)0.05).LBP50,LBP 100,LBP25 + CUR and LBP50 + cur enhanced HDAC3 activity in the presence of glucose intervention(P(27)0.05).(4)In β-TC6 cells without glucose intervention,LBP and CUR could promote the protein expression levels of Ins,Islet-1 and Pdx-1(P(27)0.05).CUR combined with LBP increased the protein expression levels of INS,Islet-1 and Pdx-1(P(27)0.05).Our results showed that LBP increased the protein expression of insulin secretion related factors.(5)Chip results showed that CUR up-regulated the H3 acetylation level of INS,Islet-1 and Pdx-1 promoters(P(27)0.05),while LBP down regulated the H3 acetylation level of Inss,Islet-1 and Pdx-1 promoters.LBP combined with cur also upregulated the H3 acetylation level of Ins,Islet-1 and Pdx-1 promoters(P(27)0.05).Combined with the above results,it is suggested that LBP has no relationship with the inhibition of HDAC3.It may increase the expression of insulin related factors Ins,Islet-1 and Pdx-1 by increasing the transcription of existing RNA.Conclusion 1.The regulation of Lycium barbarum polysaccharide on T2 DM immune response is related to the down regulation of HDAC3 gene expression.2.The mechanism of LBP promoting insulin secretion in type 2 diabetic rats may not be related to the inhibition of HDAC3 gene expression. |
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