基于HMGB1-TLR2/TLR4-NF-κB通路探索氧化苦参碱对小鼠微小隐孢子虫感染的影响

Effect of oxymatrine on Cryptosporidium parvum infection in mice based on the HMGB1-TLR2/TLR4-NF-κB pathway

目的 探讨高迁移率族蛋白B1(high mobility group box protein B1,HMGB1)-Toll样受体2(Toll-like receptor 2,TLR2)/TLR4-核因子κB(nuclear factorκB,NF-κB)通路在微小隐孢子虫感染致肠黏膜损伤中的作用,以及氧化苦参碱(oxymatrine,OMT)对小鼠微小隐孢子虫感染的干预作用。方法 4周龄SPF级BALB/c小鼠40只随机分成4组,分别为对照组、感染组、甘草酸(glycyrrhizin,GA)组及OMT组。感染组、GA组及OMT组小鼠给予地塞米松免疫抑制1周后,每只灌胃1×10^(5)个微小隐孢子虫卵囊建立微小隐孢子虫肠道感染小鼠模型。模型建立成功后,GA组小鼠连续2周腹腔注射GA 25.9 mL/(kg·d),OMT组连续2周经口灌胃OMT 50 mg/(kg·d);对照组正常饮食、饮水。治疗2周后剖杀各组小鼠,取空肠近端组织。采用苏木精-伊红(hematoxylin-eosin,HE)染色观察小鼠肠黏膜病理变化,测量肠绒毛高度、肠隐窝深度及两者比值;采用免疫组织化学染色检测小鼠肠上皮细胞中闭合蛋白(occludin)和紧密粘连蛋白1(zonula occludens protein 1,ZO1)表达水平,采用实时荧光定量PCR(quantitative real-time PCR,qPCR)检测小鼠空肠组织中HMGB1、TLR2、TLR4、髓样分化因子88(myeloid differentiation primary response gene 88,MyD88)、NF-κB p65 mRNA相对表达量。结果 HE染色结果显示,与对照组比较,感染组小鼠肠绒毛明显萎缩变短、脱落,黏膜下层水肿;GA组和OMT组小鼠肠绒毛结构趋于完整,排列趋于整齐。各组小鼠肠绒毛高度(F=6.207,P=0.000 5)、肠隐窝深度(F=6.903,P=0.000 3)及两者比值(F=37.190,P <0.000 1)差异均有统计学意义。感染组小鼠肠绒毛高度[(321.9±41.1)μm]显著低于对照组[(399.5±30.9)μm](t=4.178,P <0.01)和GA组[(383.7±42.7)μm](t=3.130,P <0.01),感染组小鼠肠隐窝深度[(185.0±35.9)μm]显著高于对照组[(128.4±23.6)μm](t=3.877,P <0.01)及GA组[(143.3±24.7)μm](t=2.710,P <0.05)。OMT组小鼠肠绒毛高度[(375.3±22.9)μm]显著高于感染组(t=3.888,P <0.01),与对照组差异无统计学意义(t=1.989,P> 0.05);OMT组小鼠肠隐窝深度[(121.5±27.3)μm]显著低于感染组[(185.0±35.9)μm](t=4.133,P <0.01),与对照组差异无统计学意义(t=0.575,P> 0.05)。感染组小鼠肠绒毛高度与肠隐窝深度比值[(1.8±0.2)]显著低于对照组[(3.1±0.3)](t=10.540,P <0.01)及GA组[(2.7±0.3)](t=7.370,P <0.01);OMT组小鼠肠绒毛高度与肠隐窝深度比值[(3.1±0.2)]显著高于感染组(t=15.020,P <0.01),与对照组差异无统计学意义(t=0.404,P> 0.05)。免疫组织化学染色结果显示,各组小鼠肠上皮细胞中occludin(F=28.031,P <0.000 1)及ZO1表达水平差异均有统计学意义(F=14.122,P <0.000 1)。感染组小鼠肠上皮细胞中occludin阳性表达率[(14.3±4.5)%]低于对照组[(28.3±0.5)%](t=3.810,P <0.01),GA组[(30.3±1.3)%]、OMT组小鼠肠上皮细胞中occludin阳性表达率[(25.8±1.5)%]显著高于感染组(t=7.620、5.391,P均<0.01),但GA组、OMT组小鼠肠上皮细胞中occludin阳性表达率与对照组差异均无统计学意义(t=1.791、2.033,P均> 0.05)。感染组小鼠肠上皮细胞中ZO1阳性表达率[(14.4±1.8)%]显著低于对照组[(24.2±2.8)%](t=4.485,P <0.01),GA组[(24.1±2.3)%](t=5.159,P <0.01)、OMT组小鼠肠上皮细胞中ZO1阳性表达率[(22.5±1.9)%]显著高于感染组(t=4.441,P <0.05),但GA组、OMT组小鼠肠上皮细胞中ZO1阳性表达率与对照组差异均无统计学意义(t=0.037、0.742,P均> 0.05)。qPCR检测结果显示,各组小鼠空肠组织中HMGB1(F=21.980,P <0.000 1)、TLR2(F=20.630,P <0.000 1)、TLR4(F=17.000,P=0.000 6)、MyD88(F=8.907,P=0.000 5)、NF-κB p65 mRNA表达水平差异均有统计学意义(F=8.889,P=0.000 7)。感染组小鼠空肠组织中HMGB1[(5.97±1.07)vs.(1.05±0.07);t=6.482,P <0.05]、TLR2[(5.92±1.29)vs.(1.10±0.14);t=5.272,P <0.05]、TLR4[(5.96±1.50)vs.(1.02±0.03);t=4.644,P <0.05]、MyD88[(3.00±1.26)vs.(1.02±0.05);t=2.734,P <0.05]、NF-κB p65 mRNA相对表达量[(2.33±0.72)vs.(1.04±0.06);t=2.665,P <0.05]均显著高于对照组。与对照组比较,GA组小鼠空肠组织中HMGB1(0.63±0.01)、TLR2(0.42±0.10)、TLR4(0.35±0.07)、MyD88(0.70±0.11)、NF-κB p65 mRNA相对表达量(0.75±0.01)均显著下降(t=8.629、5.830、11.500、4.729、6.898,P均<0.05)。与感染组比较,GA组小鼠空肠组织中MGB1、TLR2、TLR4、MyD88、NF-κB p65 mRNA相对表达量均显著降低(t=7.052、6.035、4.084、3.165、3.274,P均<0.05);OMT组小鼠空肠组织中HMGB1(1.14±0.60)、TLR2(1.00±0.24)、TLR4(1.14±0.07)、MyD88(0.96±0.25)、NF-κB p65 mRNA相对表达量(1.12±0.17)亦显著低于感染组(t=7.059、5.320、3.510、3.466、3.273,P均<0.05)。OMT组与对照组小鼠空肠组织中HMGB1、TLR2、TLR4、MyD88、NF-κB p65 mRNA相对表达量差异均无统计学意义(t=0.239、0.518、1.887、0.427、0.641,P均> 0.05)。结论 微小隐孢子虫感染小鼠后通过上调HMGB1-TLR2/TLR4-NF-κB通路表达引起肠道炎症反应、破坏肠黏膜屏障。OMT可能通过抑制HMGB1-TLR2/TLR4-NF-κB通路活性抑制小鼠肠道炎症,并修复肠黏膜屏障。

Objective To investigate the involvement of the high mobility group box protein B1 (HMGB1)-Toll-like receptor 2(TLR2)/TLR4-nuclear factorκB (NF-κB) pathway in the intestinal mucosal injury induced by Cryptosporidium parvum infection,and to examine the effect of oxymatrine (OMT) on C.parvum infection in mice.Methods Forty SPF 4-week-old BALB/c mice were randomly divided into four groups,including the control group,infection group,glycyrrhizin (GA) group and OMT group.Each mouse was orally administered with 1×10~5C.parvum oocysts one week in the infection,GA and OMT groups following dexamethasone-induced immunosuppression to model C.parvum intestinal infections in mice.Upon successful modeling,mice in the GA group were intraperitoneally injected with GA at a daily dose of 25.9 mL/kg for successive two weeks,and animals in the OMT group were orally administered OMT at a daily dose of 50 mg/kg for successive two weeks,while mice in the control group were given normal food and water.All mice were sacrificed two weeks post-treatment,and proximal jejunal tissues were sampled.The pathological changes of mouse intestinal mucosal specimens were observed using hematoxylin-eosin (HE) staining,and the mouse intestinal villous height,intestinal crypt depth and the ratio of intestinal villous height to intestinal crypt depth were measured.The occludin and zonula occludens protein 1 (ZO1) expression was determined in mouse intestinal epithelial cells using immunohistochemistry,and the relative expression of HMGB1,TLR2,TLR4,myeloid differentiation primary response gene 88(MyD88) and NF-κB p65 mRNA was quantified in mouse jejunal tissues using quantitative real-time PCR (qPCR) assay.Results HE staining showed that the mouse intestinal villi were obviously atrophic,shortened,and detached,and the submucosal layer of the mouse intestine was edematous in the infection group as compared with the control group,while the mouse intestinal villi tended to be structurally intact and neatly arranged in the GA and OMT groups.There were significant differences among the four groups in terms of the mouse intestinal villous height (F=6.207,P=0.000 5),intestinal crypt depth (F=6.903,P=0.000 3)and the ratio of intestinal villous height to intestinal crypt depth (F=37.190,P<0.000 1).The mouse intestinal villous height was lower in the infection group than in the control group[(321.9±41.1)μm vs.(399.5±30.9)μm;t=4.178,P<0.01]and the GA group[(321.9±41.1)μm vs.(383.7±42.7)μm;t=3.130,P<0.01],and the mouse intestinal crypt depth was greater in the infection group[(185.0±35.9)μm]than in the control group[(128.4±23.6)μm](t=3.877,P<0.01) and GA group[(143.3±24.7)μm](t=2.710,P<0.05).The mouse intestinal villous height was greater in the OMT group[(375.3±22.9)μm]than in the infection group (t=3.888,P<0.01),and there was no significant difference in mouse intestinal villous height between the OMT group and the control group (t=1.989,P>0.05).The mouse intestinal crypt depth was significantly lower in the OMT group[(121.5±27.3)μm]than in the infection group (t=4.133,P<0.01),and there was no significant difference in mouse intestinal crypt depth between the OMT group and the control group (t=0.575,P>0.05).The ratio of the mouse intestinal villous height to intestinal crypt depth was significantly lower in the infection group (1.8±0.2) than in the control group (3.1±0.3)(t=10.540,P<0.01) and the GA group (2.7±0.3)(t=7.370,P<0.01),and the ratio of the mouse intestinal villous height to intestinal crypt depth was significantly higher in the OMT group (3.1±0.2) than in the infection group (t=15.020,P<0.01);however,there was no significant difference in the ratio of the mouse intestinal villous height to intestinal crypt depth between the OMT group and the control group (t=0.404,P>0.05).Immunohistochemical staining showed significant differences among the four groups in terms of occludin (F=28.031,P<0.000 1) and ZO1 expression (F=14.122,P<0.000 1) in mouse intestinal epithelial cells.The proportion of positive occluding expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group[(14.3±4.5)%vs.(28.3±0.5)%;t=3.810,P<0.01],and the proportions of positive occluding expression were significantly higher in mouse intestinal epithelial cells in the GA group[(30.3±1.3)%]and OMT group[(25.8±1.5)%]than in the infection group (t=7.620 and 5.391,both P values<0.01);however,there was no significant differences in the proportion of positive occluding expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group (t=1.791 and 2.033,both P values>0.05).The proportion of positive ZO1 expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group[(14.4±1.8)%vs.(24.2±2.8)%;t=4.485,P<0.01],and the proportions of positive ZO1 expression were significantly higher in mouse intestinal epithelial cells in the GA group[(24.1±2.3)%](t=5.159,P<0.01) and OMT group than in the infection group[(22.5±1.9)%](t=4.441,P<0.05);however,there were no significant differences in the proportion of positive ZO1 expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group (t=0.037 and 0.742,both P values>0.05).qPCR assay showed significant differences among the four groups in terms of HMGB1 (F=21.980,P<0.000 1),TLR2 (F=20.630,P<0.000 1),TLR4 (F=17.000,P=0.000 6),MyD88 (F=8.907,P=0.000 5) and NF-κB p65 mRNA expression in mouse jejunal tissues (F=8.889,P=0.000 7).The relative expression of HMGB1[(5.97±1.07) vs.(1.05±0.07);t=6.482,P<0.05]、TLR2[(5.92±1.29) vs.(1.10±0.14);t=5.272,P<0.05]、TLR4[(5.96±1.50) vs.(1.02±0.03);t=4.644,P<0.05]、MyD88[(3.00±1.26) vs.(1.02±0.05);t=2.734,P<0.05]and NF-κB p65 mRNA[(2.33±0.72) vs.(1.04±0.06);t=2.665,P<0.05]was all significantly higher in mouse jejunal tissues in the infection group than in the control group.A significant reduction was detected in the relative expression of HMGB1 (0.63±0.01),TLR2 (0.42±0.10),TLR4 (0.35±0.07),MyD88 (0.70±0.11) and NF-κB p65 mRNA (0.75±0.01) in mouse jejunal tissues in the GA group relative to the control group (t=8.629,5.830,11.500,4.729 and 6.898,all P values<0.05),and the relative expression of HMGB1,TLR2,TLR4,MyD88 and NF-κB p65 mRNA significantly reduced in mouse jejunal tissues in the GA group as compared to the infection group (t=7.052,6.035,4.084,3.165 and 3.274,all P values<0.05).In addition,the relative expression of HMGB1 (1.14±0.60),TLR2 (1.00±0.24),TLR4 (1.14±0.07),MyD88 (0.96±0.25) and NF-κB p65 m RNA (1.12±0.17) was significantly lower in mouse jejunal tissues in the OMT group than in the infection group (t=7.059,5.320,3.510,3.466 and 3.273,all P values<0.05);however,there were no significant differences between the OMT and control groups in terms of relative expression of HMGB1,TLR2,TLR4,MyD88 or NF-κB p65 mRNA in mouse jejunal tissues (t=0.239,0.518,1.887,0.427 and 0.641,all P values>0.05).Conclusions C.parvuminfection causes intestinal inflammatory responses and destruction of intestinal mucosal barrier through up-regulating of the HMGB1-TLR2/TLR4-NF-κB pathway.OMT may suppress the intestinal inflammation and repair the intestinal mucosal barrier through inhibiting the activity of the HMGB1-TLR2/TLR4-NF-κB pathway.