电针对慢性溃疡性结肠炎小鼠C3a/C3aR通路相关蛋白的影响

Effects of electroacupuncture on the C3a/C3aR pathway-related proteins in mice with chronic ulcerative colitis

目的观察电针对慢性溃疡性结肠炎(ulcerative colitis,UC)模型小鼠补体C3蛋白3a(complement 3a,C3a)/补体C3蛋白3a受体(complement 3a receptor,C3aR)通路相关蛋白的影响。方法将48只BALB/c小鼠采用随机数表法分为空白组(12只)和造模组(36只)。造模组采用3%葡聚糖硫酸钠(dextran sodium sulfate,DSS)自由饮水(7 d诱导+5 d缓解)为1个周期,共4个周期构建慢性UC模型。造模成功后再二次随机分为模型组、电针组、柳氮磺胺嘧啶(sulfasalazine,SASP)组,每组12只。空白组不予处理,模型组仅捆绑,电针组针刺关元、天枢、足三里、上巨虚穴,每天20 min,SASP组每天灌胃SASP混悬液[500 mg/(kg·d)],连续14 d。记录各组小鼠的疾病活动指数(disease activity index,DAI)、体质量、结肠长度和结肠指数;HE染色观察结肠黏膜损伤指数(colonic mucosal damage index,CMDI)评分变化;Western blot检测结肠组织中C3aR蛋白的表达量;免疫组织化学染色检测半胱氨酸蛋白酶-1(caspase-1)和消皮素D(gasdermin D,GSDMD)蛋白水平;ELISA检测结肠组织中C3a蛋白及血清中肿瘤坏死因子(tumor necrosis factor,TNF-α)和白细胞介素-1β(interleukin-1β,IL-1β)等炎性因子的表达情况。结果与空白组相比,模型组小鼠的体质量显著下降,DAI评分、结肠缩短、结肠指数和CMDI评分均显著增加(P<0.01);结肠组织中C3a、C3aR蛋白的表达增加(P<0.05);Caspase-1、GSDMD蛋白的表达水平显著升高(P<0.01);血清中的IL-1β和TNF-α炎性因子的含量显著上升(P<0.01)。与模型组相比,电针组和SASP组小鼠的体质量增加,DAI和CMDI评分均降低(P<0.05),结肠长度恢复、结肠指数评分显著降低(P<0.01);结肠组织中C3aR蛋白的表达显著减少(P<0.01),C3a蛋白的表达减少(P<0.05);Caspase-1和GSDMD蛋白的表达水平降低(P<0.05);血清中IL-1β和TNF-α炎性因子的含量下降(P<0.05)。结论电针能够改善UC模型小鼠结肠组织细胞焦亡与慢性炎症,其机制可能与抑制C3a/C3aR通路蛋白的表达有关。

Objective To observe the effects of electroacupuncture on complement 3a(C3a)/complement 3a receptor(C3aR)pathway-related proteins in a mouse model of chronic ulcerative colitis(UC).Methods A total of 48 BALB/c mice were divided into a blank group(n=12)and a modeling group(n=36)by a random number table method.With a regimen of 3%dextran sodium sulfate(DSS)administered through free water drinking(seven days of induction+five days of remission)as one cycle,the modeling group underwent a total of four cycles to establish a chronic UC model,and was then randomly subdivided into model,electroacupuntcture,and sulfasalazine(SASP)groups after successful modeling,with 12 mice in each group.The blank group was not treated;the model group was only bundled;the electroacupuntcture group was treated with electroacupuntcture at Guanyuan(CV 4),Tianshu(ST 25),Zusanli(ST 36),and Shangjuxu(ST 37)for 20 minutes daily;the SASP group was given SASP suspension[500 mg/(kg·d)]by gavage daily.All intervention lasted for 14 consecutive days.The disease activity index(DAI),body weight,colon length,and colon index of each group of mice were recorded;the changes in colonic mucosal damage index(CMDI)were observed through HE staining;the expression of C3aR protein in colon tissues was examined through Western blot;the protein levels of Caspase-1 and gasdermin D(GSDMD)were checked through immunohistochemistry;the expressions of C3a protein in colon tissues and the inflammatory factors such as tumor necrosis factor(TNF-α)and interleukin-1β(IL-1β)in serum were determined through ELISA.Results Compared with the blank group,mice in the model group showed significant decreases in body weight and colon length,along with significant increases in DAI score,colon index,and CMDI score(P<0.01);the expression levels of C3a and C3aR proteins in colon tissues increased(P<0.05);the expression levels of Caspase-1 and GSDMD proteins increased significantly(P<0.01);the serum levels of IL-1βand TNF-αincreased significantly(P<0.01).Compared with the model group,mice in both electroacupuncture and SASP groups showed an increase in body weight,along with decreases in the DAI and CMDI scores(P<0.05);colon length was partially restored and the colon index score was significantly reduced in these groups(P<0.01);the expression of C3aR protein in colon tissues decreased significantly(P<0.01),and the expression of C3a protein decreased(P<0.05);the expression levels of Caspase-1 and GSDMD proteins decreased(P<0.05);the serum content of IL-1βand TNF-αdecreased(P<0.05).Conclusion Electroacupuncture can alleviate pyroptosis and chronic inflammation in colon tissues of UC model mice,and its mechanism may be related to the inhibition of the expressions of C3a/C3aR pathway-related proteins.