本文選題：ALR + 缺血再灌注損傷�。� 參考：《重慶醫(yī)科大學》2014年博士論文

【摘要】：背景與目的： 缺血再灌注損傷（IRI）引起的急性腎損傷（AKI）是臨床常見疾病，其發(fā)病率和死亡率較高，現仍缺乏有效的防治方法。近年來天然免疫反應介導的炎癥級聯反應是研究腎IRI發(fā)病機制的熱點之一。大量研究已經證實，Toll樣受體（TLR）4所介導的天然免疫反應在腎臟缺血再灌注損傷（IRI）的病理生理過程中發(fā)揮重要作用。腎臟缺血后，大量的內源性配體從損傷的腎實質細胞中釋放，與TLR4受體結合后活化下游信號通路，產生促炎細胞因子和趨化因子，介導IRI后炎癥反應。阻斷TLR4信號通路的激活對急性缺血性腎損傷有明顯的改善作用。 肝再生增強因子（ALR）是一種能促進肝細胞再生的細胞因子，我們前期研究發(fā)現ALR在腎皮質、髓質小管區(qū)高表達，外源性加入ALR能抑制腎小管上皮細胞凋亡，促進其增殖，對腎組織起保護作用。近來，研究發(fā)現ALR在肝臟有免疫調控作用，最新的體外活性研究進一步揭示ALR對激活的免疫細胞具有很強的免疫抑制作用。那么在腎IRI中，ALR對腎臟的這種保護作用是否通過其對免疫炎癥反應調節(jié)作用實現，目前尚不清楚。 在本研究中，我們首先擬通過體外實驗，采用大鼠近端腎小管上皮細胞（NRK-52E細胞）缺氧復氧模型模擬腎臟IRI過程，觀察加入ALR對腎小管上皮細胞TLR4/NF-κB信號通路的影響，繼而進一步通過體內試驗深入探討ALR對TLR4信號通路的作用機制，明確在缺血性AKI時，ALR通過抑制腎臟免疫炎癥反應從而減輕腎臟損傷，保護腎臟功能。 方法： 體外研究： 1．將體外培養(yǎng)的NRK-52E分成對照組、模型組、不同濃度重組大鼠ALR(rrALR)干預組（25ug/ml，50ug/ml），采用缺氧復氧的方法，模擬IRI過程。分別在缺氧復氧6h/12h,24h和72h時，收集細胞檢測； 2．采用RT-PCR和Western blot檢測TLR4和NF-κB mRNA和蛋白的表達 3．ELISA檢測炎癥細胞因子IL-6，IL-1β蛋白表達水平。 體內研究： 1．將雄性大鼠隨機分成假手術組、模型組、不同濃度重組人ALR（rhALR）干預組（100μg/kg,200μg/kg），采用雙側夾閉腎蒂60min，模擬IRI過程。分別在再灌注后24h,48h和72h處死大鼠，收集標本檢測。 2．采用常規(guī)生化法檢測各組大鼠血尿素氮和肌酐的差異；HE染色和TUNEL法檢測各組大鼠腎組織病理學變化和腎小管細胞凋亡情況； 3．免疫組化法檢測中性粒細胞和巨噬細胞在各組大鼠腎組織中的浸潤程度； 4．ELISA法檢測各組大鼠腎組織促炎細胞因子和趨化因子蛋白表達水平； 5．real-time PCR和免疫組化法檢測各組大鼠腎組織TLR4和其內源性配體的表達差異； 6．western blot，real-time PCR和免疫組化檢測各組大鼠TLR4下游信號分子MAPKs和NF-κB的表達變化。 結果： 體外研究： 1．NRK-52E細胞缺氧復氧后， TLR4和NF-κB mRNA和蛋白表達明顯上調，，rrALR干預后可明顯下調TLR4和NF-κB的mRNA和蛋白表達程度，rrALR高劑量處理組組較低劑量處理組組TLR4和NF-κB的表達水平下調更明顯（P0.05）。 2．ELISA法檢測顯示對照組IL-6，IL-1β蛋白低水平表達，細胞缺氧復氧后12h其表達明顯增加，外源性加入rrALR呈濃度依賴性下調IL-6，IL-1β蛋白表達水平（P0.05）。 體內研究： 1．I/R+saline組大鼠的血肌酐和尿素氮在術后24h開始明顯上升，之后逐漸下降（P0.05）；I/R+rhALR處理組的血肌酐和尿素氮較模型組明顯下降，rhALR高劑量組(I/R+rhALR2)較rhALR低劑量組(I/R+rhALR1)腎功能改善更明顯（P0.05）。 2．HE染色提示,模型組大鼠術后24h在皮質區(qū)和外髓質區(qū)可以看見典型的腎小管壞死，包括腎小管上皮細胞嚴重變性、崩解和脫落，腎小管管腔擴張，腎間質水腫，炎癥細胞浸潤；rhALR處理組的腎組織損傷程度均較模型組有所減輕，rhALR2處理組較rhALR1處理組病理改善更明顯，與腎臟損傷半定量評分結果一致（P0.05）。 3.假手術組腎組織中僅見到極其少量的TUNEL陽性細胞數，模型組組大鼠術后24h,48h和72h見到大量TUNEL陽性細胞數，主要分布在腎臟的皮質區(qū)和外髓質區(qū)，在24h時TUNEL陽性細胞數最多，rhALR處理組的TUNEL陽性細胞數在上述三個時間點均較模型組明顯減少，rhALR高劑量處理組的TUNEL陽性細胞數較rhALR低劑量處理組明顯減少（P0.05）。 4．免疫組化結果顯示假手術組僅有少量中性粒細胞和巨噬細胞，大鼠腎臟缺氧再灌注后可見大量中性粒細胞和巨噬細胞浸潤腎間質，其中中性粒細胞在再灌注后24h達最高峰，巨噬細胞在再灌注后72h達峰值。rhALR處理組在各時間點均能明顯緩解中性粒細胞和巨噬細胞的浸潤程度，并呈濃度依賴性（P0.05）。 5．real-time PCR和ELISA提示腎臟缺血再灌注損傷后，腎組織中IL-1β、IL-6和TNF-α、MCP-1和MIP-2mRNA和蛋白表達水平明顯上調（P0.05）；外源性加入rhALR后，上訴炎性細胞因子和趨化因子表達明顯減少，其中rhALR高劑量處理組的表達水平低于rhALR低劑量處理組（P0.05）。 6．real-time PCR結果顯示模型組TLR4受體及其內源性配體（HMGB1、biglycan、HAS1、HAS2、HAS3）mRNA表達在術后24小時即明顯上升（P0.05）；rhALR高劑量處理組和低劑量處理組均能明顯下調其mRNA表達水平，并呈濃度依賴性（P0.05）。免疫組化結果顯示TLR4在術后明顯增多，主要分布在腎小管上皮細胞和浸潤的炎性細胞上（P0.05）；外源性加入rhALR能明顯減少TLR4在腎組織的表達水平，rhALR高劑量處理組較低劑量處理組TLR4蛋白表達量更少（P0.05）。 7. western blot顯示大鼠在術后，腎組織中pERK/ERK,pJNK/JNK,pp38/p38均明顯升高，而rhALR處理組能明顯降低ERK、JNK、p38的磷酸化水平（P0.05）。 real-time PCR和免疫組化結果提示與假手術組相比，模型組腎組織NF-κB核酸和蛋白表達水平均明顯上升，rhALR高劑量處理組和低劑量處理組均能顯著降低NF-κB核酸和蛋白的表達水平（P0.05）。 結論： 體外研究： rrALR對NRK-52E細胞TLR4致炎信號通路的負性調節(jié)作用，可能是通過干預TLR4表達，抑制NF-κB激活，從而減少IL-1β、IL-6炎性因子的表達。 體內研究： 1．對缺血再灌注腎損傷大鼠外源性腹腔注射rhALR后，腎功能明顯改善，腎臟病理損害減輕，腎小管上皮細胞凋亡減少，并具有劑量依賴性，顯示rhALR對急性缺血性腎損傷有保護作用。 2．rhALR對急性缺血性腎損傷的保護作用與減輕腎組織炎癥反應有關。rhALR可能通過減少炎癥細胞的募集和促炎細胞因子的合成，減輕腎組織炎癥反應。 3．rhALR對腎IRI的這種炎癥抑制作用是通過下調TLR4內源性配體mRNA表達水平，抑制TLR4的活化，進而阻斷TLR4下游信號分子ERK、JNK、p38和NF-κB信號分子的激活實現的。 綜合上述兩部分的研究我們得出結論：ALR對缺血性AKI過程中的免疫炎癥反應呈抑制作用，而這種調控作用可能是通過抑制TLR4信號通路的活化，進而抑制TLR4信號轉導通路中MAPKs和NF-κB的激活，減少下游炎性細胞因子IL-6、TNF-α、IL-1β和趨化因子MCP-1和MIP-2的產生。ALR在腎IRI中對腎組織的免疫炎癥調控作用，為缺血性急性腎臟損傷提供了新的治療方向。
[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 - 偽 , 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 - 偽 , IL - 1尾 and MCP - 1 and MIP - 2 .
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R692

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