中药火把花根对油酸致急性肺损伤大鼠气道紧密连接蛋白表达的影响

Effects of traditional Chinese medicine colquhounia root tablet on the expression of tight junction protein claudin-2 and ZO-1 in bronchial epithelium tissue of rats with acute lung injury induced by oleic acid

【摘要】目的观察中药火把花根对急性肺损伤（ALI）大鼠支气管上皮紧密连接蛋白claudin-2和ZO-1表达的影响，探讨火把花根对ALI的保护机制。方法将24只健康成年清洁级雄性SD大鼠按随机数字表法分为对照组、ALI组、火把花根预处理组，每组8只。经尾静脉注射油酸0．04mL／kg复制大鼠ALI模型，对照组注射等量生理盐水（NS）；火把花根预处理组于制模前10d连续灌胃药物600mg·kg^-1·d^-1（2mL），对照组和ALI组灌胃等量NS。制模后4h取腹主动脉血、收集支气管肺泡灌洗液（BALF），测定血浆及BALF中蛋白含量，计算肺通透性指数（LPI）；处死大鼠取肺组织，测定肺湿／干质量（W／D）比值，苏木素-伊红（HE）染色后光镜下观察肺组织病理学变化，并进行肺损伤评分（LIS）；应用免疫组化法观察支气管黏膜上皮claudin-2和ZO-1的阳性表达及定位；应用蛋白质免疫印迹试验（WesternBlot）检测支气管黏膜上皮claudin-2和ZO-1的蛋白表达。结果镜下显示：与对照组比较，ALI组大鼠肺组织损伤明显，细胞水肿，细胞间连接结构紊乱，LIS、W／D及LPI均显著升高[LIS（分）：3．81±0．42比0．40±0．08，W／D：7．68±0．64比4．44±0．39，LPI：0．89±0．15比0．38±0．05，均P〈0．01]；claudin-2和ZO-1主要表达在支气管上皮细胞，且ALI组表达强度较对照组明显减弱；WesternBlot显示，ALI组claudin-2和ZO-1的蛋白表达较对照组显著下调[claudin-2蛋白（灰度值）：0．43±0．31比2．16±1．33，ZO-1蛋白（灰度值）：1．25±0．41比2．82±0．76，均P〈0．01]。与ALI组比较，火把花根预处理可明显改善ALI大鼠肺损伤程度[LIS（分）：1．22±0．39比3．81±0．42，W／D：4．62±0．84比7．68±0．64，LPI：0．46±0．07比0．89±0．15，均P〈0．01]，且elaudin-2和ZO-1的蛋白表达均明显上调[elaudin-2蛋白（灰度值）：2．98±0．91比0．43±0．31，ZO-1蛋白（灰度值）：2．35±0．51比1．25±0．41，均P〈0．01]。结论火把花根可能通过上调支气管上皮细胞紧密连接蛋白claudin-2及ZO-1的表达，以减轻ALI时气道上皮损伤，从而起到肺脏的保护性作用。

Objective To investigate the effects of traditional Chinese medicine colquhounia root tablet on the expression of tight junction protein claudin-2 and ZO-1 in bronchial epithelium tissues of rats with acute lung injury （ALI）, and to study the mechanism of protective effect of colquhounia root tablet on ALL Methods Twenty-four healthy male Sprague-Dawley （SD） rats were randomly divided into control group, ALI group and colquhounia root tablet pretreatment group, with 8 rats in each group. The model of ALI was reproduced by intravenous injection of oleie acid 0.04 mL/kg, and the rats in control group were given the same amount of normal saline （NS） instead. The rats in eolquhounia root tablet pretreatment group were intragastric administrated with colquhounia root tablet of 600 mg·kg^-1·d^-1 （2 mL） for 10 days before model reproduction, and the rats in control group and ALI group were given the same amount of NS. At 4 hours after model reproduction, the blood was drawn from abdominal aorta, and bronchoalveolar lavage fluid （BALF） was collected for determination of protein content in plasma and BALF, and the lung permeability index （LPI） was calculated. The rats were sacrificed to collect lung tissues for determination of lung wet/dry weight ratio （W/D）, the changes in pathology of lung tissue were observed after hcmatoxylin and eosin （HE） staining with light microscope, and lung injury score （LIS） was evaluated. The immunohistochemical staining was used to detect the expression and localization of claudin-2 and ZO-1 in bronchial epithelium tissues. The protein expressions of claudin-2 and ZO-1 in bronchial epithelium tissues were determined by Western Blot. Results Compared with control group, the lung injury in ALI group was more obvious including cellular edema and structural disorder of intercellular connection by optical microscope, and LIS, W/D ratio, and LPI were significantly increased （LIS： 3.81 ± 0.42 vs. 0.40 ± 0.08, W/D： 7.68 ± 0.64 vs. 4.44 ± 0.39, LPh 0.89 ± 0.15 vs. 0.38 ± 0.05, all P 〈 0.01）. Claudin-2 and ZO-1 were mainly expressed in the bronchial epithelium cell, and the expression degrees were significantly weakened in ALI group as compared with control group. It was shown by Western Blot results that compared with control group, the protein expressions of claudin-2 and ZO-1 were significantly down-regulated in ALI group [claudin-2 protein （gray value）： 0.43 ± 0.31 vs. 2.16± 1.33, ZO-1 protein （gray value）： 1.25 ± 0.41 vs. 2.82±0.76, both P 〈 0.01]. Compared with ALI group, colquhounia root pretreatment could effectively diminish the degree of ALI （LIS： 1.22±0.39 vs. 3.81 ±0.42, W/D： 4.62±0.84 vs. 7.68±0.64, LPh 0.46±0.07 vs. 0.89±0.15, all P 〈 0.01）, and the protein expressions of claudin-2 and ZO-1 were significantly up-regulated [claudin-2 protein （gray value）： 2.98 ± 0.91 vs. 0.43±0.31, ZO-1 protein （gray value）： 2.35±-0.51 vs. 1.25±-0.41, both P 〈 0.01]. Conclusion Administration of colquhounia root table could attenuate lung injury induced by oleic acid with improving epithelial barrier function via up-regulate the expression claudin-2 and ZO-1, which play a protective effect on the lung of rats with ALL .