| Objective:The purpose of this study was to explore the related factors of serum IL-1β level of type 2 diabetic patients with different diabetes duration by clinical research.Due to the fact that NLRP3 inflammasome activation is an important way of IL-1β maturation and release,the aim of the basic study is to investigate effects of the main active components of Sanhuang decoction(berberine,baicalin,emodin and rhein)on the activation of NLRP3 inflammasome in RAW264.7 cells and their mechanisms.Methods:In the clinical study,205 patients with type 2 diabetes,including 131 males and 74 females,were collected from the department of endocrinology,Jiangsu Hospital of Traditional Chinese Medicine,in strict accordance with the inclusion and exclusion criteria.General information(name,registration number,sex,age,diabetes duration,personal history,etc.),lab results(glucose metabolism,lipid metabolism,liver and kidney function,etc.)and serum samples of the patients were collected,and the level of IL-1β in serum samples was detected by cytometric bead array.Correlation analysis,Kruskal-Wallis test and chi-square test were used to compare or analyze the related factors of serum IL-1β level in patients with different diabetes duration.In the basic research,RAW264.7 macrophages were cultured in vitro,and the cytotoxic effect of berberine,baicalin and emodin on the macrophages were detected by CCK8.The activation of NLRP3 inflammasome was induced by LPS/ATP in macrophages and the effects of berberine,baicalin,emodin and rhein treatment was observed respectively.The expression levels of NLRP3,pro-Caspase-1 and Caspase-1p20 proteins in cell lysates were detected by Western Blot,and the high through-put cell imaging was used to mark NLRP3 with TRITC in RAW264.7 cells.Results:Results of the clinical study showed that after adjusting the confounding factors such as sex,age and medication history,there was a significant negative correlation between serum IL-1βlevel and HOMA-β index in patients with type 2 diabetes.When the patients were divided into groups according to diabetes durations,there were significant differences in age,course,BMI,HOMA-IR,FCP,PCP120min,TG,Urea,eGFR and the history of antihypertensive drugs among different groups.After adjusting the confounding factors,the level of serum IL-1β in patients with a diabetes duration of 2-5 years was positively correlated with eGFR;in patients with a duration of more than 10 years,the level of serum IL-1β was positively correlated with the diabetes duration and ApoE while negatively correlated with HOMA-β.The results of basic research showed that berberine,baicalin,emodin or rhein had no obvious cytotoxic effect on RAW264.7 macrophages under the concentration of 2.5 μM,5 μM and 10 μM.The results of high through-put cell imaging and immunoblotting showed that berberine,baicalin,emodin and rhein could all inhibit the expression of NLRP3 stimulated by LPS/ATP in RAW264.7 cells.The results of Western Blot showed that emodin and rhein could also inhibit the expression of pro-Caspase-1 in RAW264.7 cells,while berberine or baicalin had no significant effect on the expression of pro-Caspase-1 protein.The results of Western Blot also showed that berberine,baicalin,emodin and rhein could all inhibit the expression of Caspase-1p20 in RAW264.7 cells.Conclusion:In type 2 diabetic patients with a diabetes duration of less than 5 years,serum IL-1βlevel is positively correlated with eGFR.It is speculated that the level of serum IL-1β in patients with type 2 diabetes mellitus may be related to glomerular ultrafiltration in the early stage of diabetic nephropathy.There is a negative correlation between serum IL-1β and HOMA-β in type 2 diabetic patients with a diabetes duration of more than 10 years,and it could be inferred that the level of serum IL-1β in patients with type 2 diabetes may be related to the progressive damage of islet β cell function.The increase of serum IL-1β level in type 2 diabetic patients with a diabetes duration of more than 10 years is also closely related to the development of diabetes duration and the increase of ApoE level,but the specific significance needs to be further explored.The four active components of Sanhuang decoction(berberine,baicalin,emodin and rhein)could effectively inhibit the activation of NLRP3 inflammasome induced by LPS/ATP in macrophages.Emodin and rhein could inhibit the expression of pro-Caspase-1 in macrophages,while berberine and baicalin had no significant effect on the expression of pro-Caspase-1 in macrophages. |
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