藿朴夏苓汤对HBZY-1细胞及DN大鼠NF-κB炎症通路的影响

Effect of Huo Pu Xia Ling Tang on NF-κB Inflammatory Signaling in HBZY-1 Cells and Diabetic Nephropathy Rats

目的研究藿朴夏苓汤(HPXLT)对体内外核转录因子-κB(NF-κB)炎症通路的影响,探讨HPXLT对糖尿病肾病(DN)的作用机制。方法体外研究:采用脂多糖(LPS)刺激大鼠肾小球系膜细胞(HBZY-1)及Hep G2细胞,观察HPXLT对细胞炎症因子包括肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)含量及p-p65蛋白表达的影响,以免疫荧光法观察Hep G2细胞的NF-кB p65转核作用。体内研究:采用腹腔注射链脲佐菌素(STZ,65 mg·kg^(-1))方法制备DN大鼠模型。将大鼠随机分为对照组,模型组,HPXLT高、中、低剂量组(2,1,0.5 g·kg^(-1))和格列本脲组(5 mg·kg^(-1)),连续灌胃给药6周。采用ELISA法测定细胞上清液或大鼠血清TNF-α、IL-1β和IL-6的水平;Western Blot法测定HBZY-1细胞或大鼠肾脏p65、p-p65、TNF-α和IL-1β的蛋白表达水平。结果体外结果:与对照组比较,LPS组的p-p65蛋白表达显著增强,炎症因子TNF-α、IL-1β和IL-6的含量显著升高(P<0.01);与LPS组比较,HPXLT各剂量组的p-p65蛋白表达水平显著下调,炎症因子TNF-α、IL-1β和IL-6的含量显著降低(P<0.05,P<0.01);免疫荧光显示LPS诱导的炎症能促进NF-кB p65的转核,而HPXLT可显著抑制NF-кB p65转核作用。体内结果:与对照组比较,模型组大鼠血清的TNF-α、IL-1β、IL-6炎症因子水平及肾脏的IL-1β、TNF-α、p-p65蛋白表达水平均显著升高(P<0.01)。与模型组比较,各给药组大鼠血清的TNF-α、IL-1β和IL-6炎症因子水平均显著降低(P<0.05,P<0.01),HPXLT高、中剂量组的IL-1β、TNF-α及p-p65蛋白表达水平均明显下调(P<0.05,P<0.01),各组之间的总NF-кB p65蛋白表达水平无明显改变,差异无统计学意义(P>0.05)。结论 HPXLT可能通过抑制NF-κB炎症通路的表达,从而降低DN肾脏的炎症。

Objective To study the effect of Huo Pu Xia Ling Tang（HPXLT） on the inflammatory pathway ofnuclear factor kappa B（NF-κB） in vivo and in vitro, and to explore the mechanism of HPXLT on diabeticnephropathy（DN）. Methods In vitro study： rat glomerular mesangial cells（HBZY-1） and Hep G2 cells werestimulated by LPS,and effect of HPXLT on the content of TNF-α,IL-1β,IL-6 and expression of p-p65 proteinwas measured, and the translocation of NF-κB p65 to the nucleus of Hep G2 cells was observed byimmunofluorescent staining. In vivo study： DN rats were prepared by intraperitoneal injection of streptozotocin（STZ,65 mg·kg^（-1））. Rats were randomly divided into control group,model group,HPXLT high,medium and lowdose groups（2,1,0.5 g·kg^（-1）） and glibenclamide group（5 mg·kg^（-1）） with correspondent treatments for 6 weeks.The levels of TNF-α,IL-1β,IL-6 in cultural media and rat serum were measured by ELISA,and the proteinexpression levels of p65,p-p65,TNF-α and IL-1β in HBZY-1 cells or rat kidneys were detected by WesternBlot. Results In vitro results：compared with the control group,the expression of p-p65 protein in LPS group wassignificantly increased and the content of inflammatory factors TNF-α,IL-1β,IL-6 in cultural media increasedsignificantly（P〈0.01）. Compared with the LPS group,the levels of p-p65 protein expression in all groups ofHPXLT decreased significantly,and the contents of TNF-α,IL-1β,IL-6 were also significantly decreased（P〈0.05,P〈0.01）. Immunofluorescence observation showed that LPS-induced inflammation promoted the translocationof NF-κB p65 to the nucleus;while HPXLT significantly inhibited NF-κB p65 translocation to the nucleus. In vivo results：compared with the control group,the levels of inflammatory factors including TNF-α,IL-1β,IL-6 in ratsera and the expression levels of IL-1β,TNF-α and p-p65 protein in the kidney were significantly increased in themodel group（P〈0.01）. Compared with the model group,the levels of TNF-α,IL-1β and IL-6 in the rat serumwere significantly decreased（P〈0.05,P〈0.01）. The expression levels of IL-1β,TNF-α and p-p65 protein inthe HPXLT high and middle dose groups were obviously decreased（P〈0.05,P〈0.01）. There was no significantdifference in the expression of the total NF-κB p65 protein among the groups（P〈0.05）. Conclusion HPXLTcould inhibit the inflammation of DN kidneys by inhibiting the expression of NF-κB inflammatory pathway.