瑞马唑仑调节TLR4/MyD88/NF-κB信号通路对烧伤大鼠肠上皮细胞凋亡的影响

doi:10.11958/20240667

摘要/Abstract

摘要：

目的探究瑞马唑仑（Rem）调节Toll样受体4（TLR4）/髓样分化因子88（MyD88）/核因子-κB（NF-κB）信号通路对烧伤大鼠肠上皮细胞凋亡的影响。方法将造模成功的烧伤大鼠随机分为模型组（Model组），药物低、中、高剂量处理组（Rem-L组、Rem-M组、Rem-H组）和高剂量瑞马唑仑+TLR4激活剂组（Rem-H+LPS组），另取健康的大鼠作对照组（Control组）。在对大鼠尾静脉采血且行安乐死后取其肠组织样本。酶联免疫吸附试验（ELISA）检测血清炎性因子白细胞介素（IL）-1β、IL-6水平；HE染色观察肠组织形态；TUNEL检测试剂盒检测细胞凋亡；免疫组化检测紧密连接蛋白ZO-1、Occludin表达；免疫印迹实验检测凋亡蛋白Bax及TLR4/MyD88/NF-κB信号通路蛋白表达。结果与Control组相比，Model组细胞排列紊乱，有炎症表现，IL-1β、IL-6水平、细胞凋亡率升高，Bax、TLR4、MyD88、p-NF-κB/NF-κB表达上调，ZO-1、Occludin表达下调（P<0.05）；与Model组比较，Rem-L、Rem-M、Rem-H组肠黏膜炎症浸润逐渐减轻，IL-1β、IL-6水平、细胞凋亡率降低，Bax、TLR4、MyD88、p-NF-κB/NF-κB表达下调，ZO-1、Occludin表达上调，呈剂量依赖性（P<0.05）；与Rem-H组相比，Rem-H+LPS组组织炎症加重，IL-1β、IL-6水平、细胞凋亡率升高，Bax、TLR4、MyD88、p-NF-κB/NF-κB表达上调，ZO-1、Occludin表达下调（P<0.05）。结论 Rem可能通过抑制TLR4/MyD88/NF-κB信号通路缓解烧伤大鼠肠上皮细胞的损伤，从而保护肠黏膜。

关键词: 烧伤, Toll样受体4, 髓样分化因子88, NF-κB, 细胞凋亡, 瑞马唑仑, 肠上皮细胞

Abstract:

Objective To investigate the effect of remimazolam (Rem) on apoptosis of intestinal epithelial cells in burned rats by regulating Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. Methods The successfully constructed burned rat model was randomly divided into the model group, the Rem low dose treatment (Rem-L) group, the Rem medium dose treatment (Rem-M) group and the Rem high dose treatment (Rem-H) group, and Rem-H+TLR4 activator (LPS) group. Healthy rats were taken as the control group. After blood samples were collected from tail vein of rats and intestinal tissue samples were taken after euthanasia. Serum levels of inflammatory cytokines interleukin-1β and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). HE staining was applied to observe the morphology of intestinal tissue. TUNEL detection kit was used to detect apoptosis. Immunohistochemistry was used to detect the expression of tight junction proteins ZO-1 and Occludin. Immunoblotting experiments were used to detect the expression levels of apoptotic proteins Bax and TLR4/MyD88/NF-κB signaling pathway proteins. Results Compared with the control group, cell arrangement was disordered with inflammation, and IL-1β and IL-6 levels and apoptosis rate were increased, expression levels of Bax, TLR4, MyD88 and p-NF-κB/NF-κB were up-regulated, and expression levels of ZO-1 and Occludin were down-regulated in the model group (P<0.05). Compared with the model group, inflammatory infiltration of intestinal mucosa was gradually reduced in the Rem-L, Rem-M and Rem-H groups, the apoptosis rate, IL-1β and IL-6 levels were decreased, the expression levels of Bax, TLR4, MyD88 and p-NF-κB/NF-κB were down-regulated, and the expression of ZO-1 and Occludin was up-regulated in a dose-dependent manner (P<0.05). Compared with the Rem-H group, the tissue inflammation was aggravated, the apoptosis rate, levels of IL-1β and IL-6 were increased, expression levels of Bax, TLR4, MyD88 and p-NF-κB/NF-κB were up-regulated, and expression levels of ZO-1 and Occludin were down-regulatedin in the Rem-H+LPS group (P<0.05). Conclusion Remimazolam may alleviate the damage of intestinal epithelial cells in burned model rats by inhibiting TLR4/MyD88/NF-κB signaling pathway, thus protecting intestinal mucosa.

Key words: burns, Toll-like receptor 4, myeloid differentiation factor 88, NF-kappa B, apoptosis, Remimazolam, intestinal epithelial cells

中图分类号:

R644

图/表 9

表1 各组大鼠血清IL-1β、IL-6水平比较（n=6，ng/L，$\bar{x} \pm s$）

Tab.1 Comparison of serum levels of IL-1β and IL-6 between the six groups

组别	IL-1β	IL-6
Control组	5.73±0.64	8.64±1.03
Model组	83.47±9.15^a	69.76±7.28^a
Rem-L组	56.83±6.42^b	43.55±5.36^b
Rem-M组	25.39±2.68^bc	29.42±3.34^bc
Rem-H组	13.97±1.82^bcd	16.53±1.90^bcd
Rem-H+LPS组	34.75±3.91^e	32.06±3.79^e
F	200.098^＊＊	150.559^＊＊

图1 肠黏膜组织病理形态（HE染色，×200）

Fig.1 Pathological morphology of intestinal mucosa (HE staining, ×200)

表2 各组大鼠肠上皮细胞凋亡率和Bax蛋白表达比较（n=6，$\bar{x} \pm s$）

Tab.2 Comparison of apoptosis rate and expression of Bax protein in intestinal epithelial cells between the six groups

组别	细胞凋亡率/%	Bax
Control组	2.68±0.31	0.32±0.03
Model组	29.67±4.12^a	1.18±0.16^a
Rem-L组	23.54±3.69^b	0.81±0.08^b
Rem-M组	15.38±2.84^bc	0.65±0.06^bc
Rem-H组	10.96±1.25^bcd	0.42±0.04^bcd
Rem-H+LPS组	19.05±2.14^e	0.74±0.07^e
F	72.424^＊＊	78.419^＊＊

图2 各组细胞凋亡情况（TUNEL染色，×200）

Fig.2 The apoptosis of cells in each group (TUNEL staining, ×200)

图3 Western blot检测Bax蛋白表达 A：Control组；B：Model组；C：Rem-L组；D：Rem-M组；E：Rem-H组；F：Rem-H+LPS组。

Fig.3 Bax protein expression detected by Western blot assay

表3 各组大鼠肠组织ZO-1、Occludin蛋白表达比较（n=6，$\bar{x} \pm s$）

Tab.3 Comparison of ZO-1 and Occludin expression in intestinal tissue of rats between the six groups

组别	ZO-1	Occludin
Control组	1.13±0.15	1.25±0.18
Model组	0.30±0.03^a	0.23±0.02^a
Rem-L组	0.53±0.05^b	0.45±0.04^b
Rem-M组	0.76±0.07^bc	0.66±0.06^bc
Rem-H组	0.94±0.11^bcd	0.89±0.09^bcd
Rem-H+LPS组	0.62±0.06^d	0.56±0.05^d
F	68.181^＊＊	94.657^＊＊

图4 免疫组化检测ZO-1、Occludin蛋白表达（×400）

Fig.4 Immunohistochemical detection of ZO-1 and Occludin protein expression (×400)

图5 Western blot检测TLR4/MyD88/NF-κB信号通路相关蛋白表达 A：Control组；B：Model组；C：Rem-L组；D：Rem-M组；E：Rem-H组；F：Rem-H+LPS组。

Fig.5 Western blot detection of the expression of TLR4/MyD88/NF-κB signaling pathway-related proteins

表4 各组大鼠肠组织TLR4、MyD88、p-NF-κB水平比较（n=6，$\bar{x} \pm s$）

Tab.4 Comparison of TLR4, MyD88 and p-NF-κB levels in intestinal tissue of rats between the six groups

组别	TLR4	MyD88	p-NF-κB/NF-κB
Control组	0.21±0.02	0.27±0.03	0.30±0.03
Model组	1.16±0.21^a	1.24±0.15^a	0.90±0.08^a
Rem-L组	0.91±0.11^b	0.82±0.08^b	0.78±0.08^b
Rem-M组	0.70±0.07^bc	0.65±0.06^bc	0.56±0.06^bc
Rem-H组	0.52±0.05^bcd	0.37±0.04^bcd	0.22±0.04^bcd
Rem-H+LPS组	0.80±0.08^e	0.72±0.07^e	0.61±0.06^e
F	54.873^＊＊	108.250^＊＊	111.899^＊＊

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