川芎嗪对脂多糖诱导肺损伤大鼠肺组织水通道蛋白1、5表达影响的研究

Effect of tetramethylpyrazine's on expressions of aquaporin1,5 in lipopolysaccharide-induced lung injury in rats

目的:初步探讨川芎嗪(tetramethylpyrazine,TMP)对脂多糖(lipopolysaccharide,LPS)诱导肺损伤大鼠肺组织水通道蛋白(aquaporins,AQPs)1、5表达的调节作用。方法:健康雄性SPF级SD大鼠32只随机分为正常对照组(C组)、TMP处理组(T组)、LPS刺激组(L组)、TMP治疗组(LT组),每组8只。L组采用尾静脉注射6 mg/kg LPS,T组给予TMP 100 mg/kg腹腔注射,LT组大鼠注射LPS 1 h后立即给予TMP 100 mg/kg腹腔注射。8 h后麻醉处死大鼠留取肺组织标本。测定肺组织湿/干重比(wet/dry ratio,W/D),苏木素-伊红(HE)染色观察肺组织病理形态,实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)检测肺组织匀浆AQP1和AQP5的m RNA水平,免疫组化染色、Western blot检测肺组织AQP1、AQP5蛋白表达。结果:肺组织切片HE染色光镜下可见C组、T组肺组织结构完整、清晰;L组肺泡结构紊乱,弥漫性肺泡间隔增厚,肺泡腔内有炎性细胞浸润、出血和水肿,而LT组上述病理表现较L组减轻。L组平均肺组织湿/干重比值(5.577±0.243)明显高于其余3组(P<0.01);与C、T 2组(3.430±0.251、3.251±0.142)相比,LT组湿/干重比值(4.384±0.236)增加,差异存在统计学意义(P<0.01)。q RT-PCR结果显示T组AQP1、AQP5的RNA水平(1.462±0.080、1.267±0.063)高于C组(1.160±0.229、0.940±0.087),同时,L组两者转录水平下调(0.607±0.203、0.139±0.046),而LT组m RNA水平(0.848±0.071、0.314±0.134)高于L组(P<0.05),免疫组化(C组:2.096±0.114、2.285±0.266;T组:3.253±0.149、3.790±0.308;L组:1.133±0.202、1.068±0.221;LT组:1.562±0.085、1.647±0.239)、Western blot结果(C组:1.443±0.103、1.278±0.061;T组:1.667±0.087、1.644±0.102;L组:0.974±0.090、0.529±0.100;LT组:1.179±0.051、0.805±0.082)与q RT-PCR一致(P<0.05)。结论:川芎嗪可有效减轻脂多糖诱导急性呼吸窘迫综合征的肺水肿,起到积极的保护作用,其机制可能与上调AQP1、AQP5的表达有关。

Objective：To preliminarily investigate the effects of tetramethylpyrazine（TMP）on expressions of aquaporin1,5 in lipopolysaccharide（LPS）-induced lung injury in rats. Methods：Thirty-two healthy male SPF grade SD rats were randomly divided into the normal control group（group C）,the TMP intervention group（group T）,the LPS stimulation group（group L） and the TMP treatment group（LT group）,with eight rats in each group. Rats in the group L were given LPS（6 mg/kg）through tail vein injection,rats in group T were given TMP（100 mg/kg）through intraperitoneal injection,and rats in group LT were given LPS for 1 h followed by TMP（100mg/kg）through intraperitoneal injection immediately. After 8 hours,all rats were anesthetized and sacrificed,with lung tissue specimens harvested. Wet/dry ratio（W/D）of the lung tissue was detected. In addition,hematoxylin-eosin（HE） staining was performed to observe the lung tissue morphology;Quantitative real-time PCR（q RT-PCR）was used to detect the AQP1 m RNA and AQP5 m RNA levels of the lung tissue homogenate. In addition,immunohistochemistry and Western blot were used to detect the protein expression of AQP1 and AQP5 in the lung tissue. Results：After HE staining of the lung tissue sections,the lung tissue was integral and clear in its structure in group C and T under light microscope. Alveoli were disorderly in structures,the diffuse alveolar septa were thickened,and alveolar inflammatory cell infiltration,hemorrhage and edema were observed in group L. While the LT group had milder pathological manifestations than group L. The average W/D ratio of the lung tissue in group L（5.577±0.243）was higher than that of each of the other three groups significantly（P〈0.01）. Compared with group C and T（3.430±0.251、3.251±0.142）,the W/D ratio in the group LT（4.384±0.236）was increased,and the difference was significant（P〈0.01）. The results of q RT-PCR showed that the levels of AQP1 RNA and AQP5 RNA in group T（1.462±0.080,1.267±0.063）were higher than those in group C（1.160±0.229,0.940±0.087）and the levels in group L（0.607±0.203,0.139±0.046）were down-regulated. Meanwhile,the levels in group LT（0.848±0.071,0.314±0.134）were higher than those in group L（P〈0.05）. The results of immunohistochemistry（C group：2.096±0.114,2.285±0.266;T group：3.253±0.149,3.790±0.308;L group：1.133±0.202,1.068 ±0.221;LT group：1.562 ±0.085,1.647±0.239）and Western blot（C group：1.443±0.103,1.278±0.061;T group：1.667±0.087,1.644±0.102;L group：0.974±0.090,0.529±0.100;LT group：1.179±0.051,0.805±0.082）were consistent with those of q RT-PCR（P〈0.05）. Conclusion：TMP can exhibit positive protective effects by mitigating LPS-induced pulmonary edema in ARDS effectively,and the mechanism may be associated with the upregulation of the expressions of AQP1 and AQP5.