肝再生增强因子在缺血性急性肾损伤中对TLR4信号通路的调控机制研究

发布时间：2018-05-18 08:18

本文选题：ALR + 缺血再灌注损伤　；参考：《重庆医科大学》2014年博士论文

【摘要】：背景与目的：缺血再灌注损伤（IRI）引起的急性肾损伤（AKI）是临床常见疾病，其发病率和死亡率较高，现仍缺乏有效的防治方法。近年来天然免疫反应介导的炎症级联反应是研究肾IRI发病机制的热点之一。大量研究已经证实，Toll样受体（TLR）4所介导的天然免疫反应在肾脏缺血再灌注损伤（IRI）的病理生理过程中发挥重要作用。肾脏缺血后，大量的内源性配体从损伤的肾实质细胞中释放，与TLR4受体结合后活化下游信号通路，产生促炎细胞因子和趋化因子，介导IRI后炎症反应。阻断TLR4信号通路的激活对急性缺血性肾损伤有明显的改善作用。肝再生增强因子（ALR）是一种能促进肝细胞再生的细胞因子，我们前期研究发现ALR在肾皮质、髓质小管区高表达，外源性加入ALR能抑制肾小管上皮细胞凋亡，促进其增殖，对肾组织起保护作用。近来，研究发现ALR在肝脏有免疫调控作用，最新的体外活性研究进一步揭示ALR对激活的免疫细胞具有很强的免疫抑制作用。那么在肾IRI中，ALR对肾脏的这种保护作用是否通过其对免疫炎症反应调节作用实现，目前尚不清楚。在本研究中，我们首先拟通过体外实验，采用大鼠近端肾小管上皮细胞（NRK-52E细胞）缺氧复氧模型模拟肾脏IRI过程，观察加入ALR对肾小管上皮细胞TLR4/NF-κB信号通路的影响，继而进一步通过体内试验深入探讨ALR对TLR4信号通路的作用机制，明确在缺血性AKI时，ALR通过抑制肾脏免疫炎症反应从而减轻肾脏损伤，保护肾脏功能。方法：体外研究： 1．将体外培养的NRK-52E分成对照组、模型组、不同浓度重组大鼠ALR(rrALR)干预组（25ug/ml，50ug/ml），采用缺氧复氧的方法，模拟IRI过程。分别在缺氧复氧6h/12h,24h和72h时，收集细胞检测； 2．采用RT-PCR和Western blot检测TLR4和NF-κB mRNA和蛋白的表达 3．ELISA检测炎症细胞因子IL-6，IL-1β蛋白表达水平。体内研究： 1．将雄性大鼠随机分成假手术组、模型组、不同浓度重组人ALR（rhALR）干预组（100μg/kg,200μg/kg），采用双侧夹闭肾蒂60min，模拟IRI过程。分别在再灌注后24h,48h和72h处死大鼠，收集标本检测。 2．采用常规生化法检测各组大鼠血尿素氮和肌酐的差异；HE染色和TUNEL法检测各组大鼠肾组织病理学变化和肾小管细胞凋亡情况； 3．免疫组化法检测中性粒细胞和巨噬细胞在各组大鼠肾组织中的浸润程度； 4．ELISA法检测各组大鼠肾组织促炎细胞因子和趋化因子蛋白表达水平； 5．real-time PCR和免疫组化法检测各组大鼠肾组织TLR4和其内源性配体的表达差异； 6．western blot，real-time PCR和免疫组化检测各组大鼠TLR4下游信号分子MAPKs和NF-κB的表达变化。结果：体外研究： 1．NRK-52E细胞缺氧复氧后， TLR4和NF-κB mRNA和蛋白表达明显上调，，rrALR干预后可明显下调TLR4和NF-κB的mRNA和蛋白表达程度，rrALR高剂量处理组组较低剂量处理组组TLR4和NF-κB的表达水平下调更明显（P0.05）。 2．ELISA法检测显示对照组IL-6，IL-1β蛋白低水平表达，细胞缺氧复氧后12h其表达明显增加，外源性加入rrALR呈浓度依赖性下调IL-6，IL-1β蛋白表达水平（P0.05）。体内研究： 1．I/R+saline组大鼠的血肌酐和尿素氮在术后24h开始明显上升，之后逐渐下降（P0.05）；I/R+rhALR处理组的血肌酐和尿素氮较模型组明显下降，rhALR高剂量组(I/R+rhALR2)较rhALR低剂量组(I/R+rhALR1)肾功能改善更明显（P0.05）。 2．HE染色提示,模型组大鼠术后24h在皮质区和外髓质区可以看见典型的肾小管坏死，包括肾小管上皮细胞严重变性、崩解和脱落，肾小管管腔扩张，肾间质水肿，炎症细胞浸润；rhALR处理组的肾组织损伤程度均较模型组有所减轻，rhALR2处理组较rhALR1处理组病理改善更明显，与肾脏损伤半定量评分结果一致（P0.05）。 3.假手术组肾组织中仅见到极其少量的TUNEL阳性细胞数，模型组组大鼠术后24h,48h和72h见到大量TUNEL阳性细胞数，主要分布在肾脏的皮质区和外髓质区，在24h时TUNEL阳性细胞数最多，rhALR处理组的TUNEL阳性细胞数在上述三个时间点均较模型组明显减少，rhALR高剂量处理组的TUNEL阳性细胞数较rhALR低剂量处理组明显减少（P0.05）。 4．免疫组化结果显示假手术组仅有少量中性粒细胞和巨噬细胞，大鼠肾脏缺氧再灌注后可见大量中性粒细胞和巨噬细胞浸润肾间质，其中中性粒细胞在再灌注后24h达最高峰，巨噬细胞在再灌注后72h达峰值。rhALR处理组在各时间点均能明显缓解中性粒细胞和巨噬细胞的浸润程度，并呈浓度依赖性（P0.05）。 5．real-time PCR和ELISA提示肾脏缺血再灌注损伤后，肾组织中IL-1β、IL-6和TNF-α、MCP-1和MIP-2mRNA和蛋白表达水平明显上调（P0.05）；外源性加入rhALR后，上诉炎性细胞因子和趋化因子表达明显减少，其中rhALR高剂量处理组的表达水平低于rhALR低剂量处理组（P0.05）。 6．real-time PCR结果显示模型组TLR4受体及其内源性配体（HMGB1、biglycan、HAS1、HAS2、HAS3）mRNA表达在术后24小时即明显上升（P0.05）；rhALR高剂量处理组和低剂量处理组均能明显下调其mRNA表达水平，并呈浓度依赖性（P0.05）。免疫组化结果显示TLR4在术后明显增多，主要分布在肾小管上皮细胞和浸润的炎性细胞上（P0.05）；外源性加入rhALR能明显减少TLR4在肾组织的表达水平，rhALR高剂量处理组较低剂量处理组TLR4蛋白表达量更少（P0.05）。 7. western blot显示大鼠在术后，肾组织中pERK/ERK,pJNK/JNK,pp38/p38均明显升高，而rhALR处理组能明显降低ERK、JNK、p38的磷酸化水平（P0.05）。 real-time PCR和免疫组化结果提示与假手术组相比，模型组肾组织NF-κB核酸和蛋白表达水平均明显上升，rhALR高剂量处理组和低剂量处理组均能显著降低NF-κB核酸和蛋白的表达水平（P0.05）。结论：体外研究： rrALR对NRK-52E细胞TLR4致炎信号通路的负性调节作用，可能是通过干预TLR4表达，抑制NF-κB激活，从而减少IL-1β、IL-6炎性因子的表达。体内研究： 1．对缺血再灌注肾损伤大鼠外源性腹腔注射rhALR后，肾功能明显改善，肾脏病理损害减轻，肾小管上皮细胞凋亡减少，并具有剂量依赖性，显示rhALR对急性缺血性肾损伤有保护作用。 2．rhALR对急性缺血性肾损伤的保护作用与减轻肾组织炎症反应有关。rhALR可能通过减少炎症细胞的募集和促炎细胞因子的合成，减轻肾组织炎症反应。 3．rhALR对肾IRI的这种炎症抑制作用是通过下调TLR4内源性配体mRNA表达水平，抑制TLR4的活化，进而阻断TLR4下游信号分子ERK、JNK、p38和NF-κB信号分子的激活实现的。综合上述两部分的研究我们得出结论：ALR对缺血性AKI过程中的免疫炎症反应呈抑制作用，而这种调控作用可能是通过抑制TLR4信号通路的活化，进而抑制TLR4信号转导通路中MAPKs和NF-κB的激活，减少下游炎性细胞因子IL-6、TNF-α、IL-1β和趋化因子MCP-1和MIP-2的产生。ALR在肾IRI中对肾组织的免疫炎症调控作用，为缺血性急性肾脏损伤提供了新的治疗方向。
[Abstract]:Background and Purpose :

Acute kidney injury ( AKI ) caused by ischemia - reperfusion injury ( IRI ) is a common disease in clinic , and its morbidity and mortality are high , and there is still a lack of effective control methods . In recent years , natural immune response mediated by Toll - like receptor ( TLR ) 4 plays an important role in the pathogenesis of renal ischemia - reperfusion injury ( IRI ) .

In recent years , it has been found that the immune regulation in the renal cortex and the small tubular region of the renal cortex can be inhibited by exogenous addition of the alr to the renal cortex .

In this study , we first intend to simulate the renal IRI by using the hypoxia - complex oxygen model of proximal tubular epithelial cells ( NRK - 52E cells ) in rats and observe the effect of the addition of alr on the signaling pathway of the renal tubular epithelial cells .

Method :

In vitro studies :

1 . The NRK - 52E cultured in vitro was divided into the control group , the model group , the recombinant rats of different concentrations ( 25 ug / ml , 50ug / ml ) , and the process of IRI was simulated by the method of oxygen - free complex oxygen , and the cells were collected at 6h / 12h , 24h and 72h respectively .

2 . RT - PCR and Western blot were used to detect the mRNA and protein expression of TL4 and NF - 魏B .

3 . ELISA was used to detect the level of IL - 6 and IL - 1尾 in inflammatory cytokines .

In vivo studies :

1 . Male rats were randomly divided into two groups : sham operation group , model group , different concentration recombinant human alr ( rhalr ) intervention group ( 100 渭g / kg , 200 渭g / kg ) . The rats were sacrificed at 24 h , 48 h and 72 h after reperfusion , and the specimens were collected .

2 . The difference of blood urea nitrogen and creatinine was detected by routine biochemistry method .
The pathological changes and apoptosis of renal tubular cells were detected by HE staining and TUNEL method .

3 . Immunohistochemical method was used to detect the infiltration of neutrophils and macrophages in renal tissue of each group .

4 . The levels of pro - inflammatory cytokines and chemokine protein expression were detected by ELISA .

5 . Real - time PCR and immunohistochemistry were used to detect the differences in the expression of TLR 4 and endogenous ligand in renal tissue of rats in each group .

6 . The expression of MAPKs and NF - 魏B was detected by western blot , real - time PCR and immunohistochemistry .

Results :

In vitro studies :

1.NRK - 52E mRNA and protein expression were up - regulated in NRK - 52E cells after hypoxia .

2 . The levels of IL - 6 and IL - 1尾 in the control group were detected by ELISA , and the expression of IL - 6 and IL - 1尾 was significantly increased at 12 h after hypoxia .

In vivo studies :

The serum creatinine and urea nitrogen in the 1 . I / R + saline group increased significantly at 24 h after operation ( P0.05 ) .
The serum creatinine and urea nitrogen in the I / R + rhALR2 treated group were significantly lower than those in the low dose group ( I / R + rhALR2 ) , and the improvement of renal function in the low dose group ( I / R + rhALR2 ) was more obvious ( P0.05 ) .

2 . HE staining showed that the necrosis of the tubular epithelial cells was seen in the cortical and external medullary regions 24h after operation , including severe degeneration , disintegration and detachment of tubular epithelial cells , dilatation of tubular lumen , interstitial edema and infiltration of inflammatory cells .
In the rhALR2 treatment group , the damage degree of the renal tissue was reduced compared with the model group , and the rhALR2 treated group had more obvious pathological improvement compared with the rhALR1 treatment group , which was consistent with the result of the semi - quantitative score of the kidney injury ( P0.05 ) .

3 . The number of TUNEL positive cells was seen in the renal tissue of the sham operation group , and the number of TUNEL positive cells was seen in the cortical and outer medulla of the kidney . The number of TUNEL - positive cells was significantly decreased at 24 h , and the number of TUNEL - positive cells in the treated group was significantly decreased at the time of 24 h . TUNEL - positive cells in the high - dose treatment group were significantly decreased compared with the low - dose group ( P0.05 ) .

4 . Immunohistochemical results showed that only a small amount of neutrophils and macrophages were found in the sham operation group , and a large number of neutrophils and macrophages infiltrated the kidney stroma after hypoxia and reperfusion in rats .

5 . Real - time PCR and ELISA suggested that the levels of IL - 1尾 , IL - 6 and TNF - 伪 , MCP - 1 and MIP - 2 mRNA and protein in renal tissue were significantly increased after renal ischemia / reperfusion injury ( P0.05 ) .
The expression level of inflammatory cytokines and chemokine expression was significantly decreased after exogenous addition of rhalr , and the level of expression in the high dose treatment group was lower than that in the low dose treatment group ( P0.05 ) .

6 . The real - time PCR results showed that the mRNA expression of the receptor and its endogenous ligand in the model group was significantly increased 24 hours after operation ( P0.05 ) .
The levels of mRNA expression and concentration - dependent ( P0.05 ) were observed in both high - dose treatment group and low - dose treatment group ( P0.05 ) .
It was found that the expression level of TL4 in renal tissue was significantly reduced by exogenous addition of rhADr , and the expression level was lower in the high - dose treatment group than in the low - dose treatment group ( P0.05 ) .

7 . Western blot showed that the levels of pERK / ERK , p38 and p38 / p38 in renal tissue were significantly higher than those in sham - operated group . The levels of NF - 魏B and protein were significantly increased in the model group compared with sham - operated group , and the expression level of NF - 魏B and protein was significantly decreased in both high - dose treatment group and low - dose treatment group ( P0.05 ) .

Conclusion :

In vitro studies :

The negative regulation of the signal pathway caused by NRK - 52E cells in NRK - 52E cells may be mediated by the intervention of IL - 1尾 , IL - 6 and IL - 6 in the expression of IL - 1尾 , IL - 6 , and the expression of IL - 1尾 and IL - 6 .

In vivo studies :

1 . The renal function was significantly improved , renal pathological injury was relieved , the apoptosis of renal tubular epithelial cells decreased , and the dose - dependent effect was shown to show the protective effect on acute ischemic kidney injury in rats with ischemia - reperfusion renal injury .

2 . The protective effect on acute ischemic kidney injury is related to the reduction of inflammatory response of renal tissue . rhalr may reduce the inflammatory response of renal tissue by reducing the recruitment of inflammatory cells and the synthesis of pro - inflammatory cytokines .

3 . The anti - inflammatory effects of rhalr on renal IRI were achieved by downregulating the level of endogenous ligand mRNA expression , inhibiting the activation of TLR , and then blocking the activation of ERK and p38 and NF - 魏B signal molecules in downstream signaling molecules .

Taken together the two parts , we concluded that the immune inflammatory response in the process of ischemic AKI was inhibited , which could inhibit the activation of MAPKs and NF - 魏B in the signal transduction pathway , reduce the production of downstream inflammatory cytokines , IL - 6 , TNF - 伪 , IL - 1尾 and MCP - 1 and MIP - 2 .
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692

【参考文献】