冷暴露对内毒素性急性肺损伤小鼠TLR4信号通路的影响

Effects of Cold Exposure on TLR4 Signaling Pathway in Mice with Endotoxin Induced Acute Lung Injury

为了研究冷暴露对脂多糖(LPS)诱导的急性肺损伤的影响及其与Toll样受体4(TLR4)信号通路的关系,本试验将40只健康BALB/c雄性小鼠,随机分为正常对照组(N组)、冷暴露组(C组)、LPS组(L组)和冷暴露+LPS组(C+L组),每组10只,其中L组和C+L组小鼠鼻滴LPS溶液[0.5 mg/(kg·bw)],N组和C组鼻滴等体积无菌PBS溶液,滴鼻后将N组和L组小鼠置于室温环境下,C组和C+L组小鼠置于(4±2)℃通风冷室中,6 h后采用BCA法检测支气管肺泡灌洗液(BALF)中总蛋白浓度和白细胞数量;ELISA法检测BALF中肿瘤坏死因子α(TNF-α)和白介素6(IL-6)含量;取肺组织称重,计算肺组织的湿干重比(W/D),苏木精-伊红(H.E.)染色法观察肺组织病理改变;Western blot法测定肺组织TLR4蛋白表达量。结果显示,与N组比较,C组总蛋白浓度、白细胞数量,TNF-α、IL-6含量,肺组织W/D及肺组织TLR4蛋白表达量各项指标均无明显变化,差异没有统计学意义(P>0.05);N组和C组肺组织H.E.染色结果均正常,没有明显的差别;与N组比较,L组上述各项指标均显著升高,差异有统计学意义(P<0.05或P<0.01);L组肺组织发生组织病理学改变,出现了炎性细胞浸润、肺水肿、肺泡出血和肺泡间隔增厚,肺泡结构受损;与C组比较,C+L组上述各项指标均显著升高,差异有统计学意义(P<0.05或P<0.01),并且肺组织发生与L组相似的组织病理学改变;与L组比较,C+L组上述各项指标均显著升高,差异有统计学意义(P<0.05),并且肺组织的组织病理学改变加重。结果表明,冷暴露可加重LPS诱导的急性肺损伤,损伤作用可能与TLR4信号通路机制有关。

To investigate the influence of cold exposure on lipopolysaccharide(LPS)-induced acute lung injury and its relationship with the Toll-like receptor 4(TLR4)signaling pathway,40 healthy male BALB/c mice were randomly divided into the Normal control group(N group),Cold exposure group(C group),LPS group(L group),and Cold exposure+LPS group(C+L group),with 10 mice in each group.Mice in the L and C+L groups received intranasal administration of LPS solution[0.5 mg/(kg·bw)],while N and C groups received an equal volume of sterile PBS.After intranasal administration,N and L group mice were kept at room temperature,while C and C+L group mice were placed in a ventilated cold chamber at(4±2)℃.After 6 hours,the bronchoalveolar lavage fluid(BALF)was collected for the determination of total protein concentration and white blood cell count using the BCA method.The levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in BALF were measured by enzyme linked immunosorbent assay(ELISA).Lung tissues were weighed to calculate the wet to dry weight ratio(W/D).Histopathological changes in lung tissues were observed using hematoxylin-eosin(H.E.)staining.Western blot analysis was employed to determine the expression of TLR4 protein in lung tissues.The results showed that compared with the N group,there were no significant changes in total protein concentration,white blood cell count,TNF-αand IL-6 levels,lung tissue W/D,and TLR4 protein expression in the C group,and the differences were not statistically significant(P>0.05).H.E.staining results of lung tissues in both N and C groups were normal,with no significant differences.In comparison with the N group,all the above indicators in the L group significantly increased,with statistically significant differences(P<0.05 or P<0.01).Pathological changes in lung tissue of the L group included inflammatory cell infiltration,pulmonary edema,alveolar hemorrhage,and thickening of alveolar septa,indicating damage to the alveolar structure.Compared with the C group,all the above indicators in the C+L group significantly increased,with statistically significant differences(P<0.05 or P<0.01),and lung tissue underwent similar pathological changes to the L group.In comparison with the L group,all the above indicators in the C+L group significantly increased,with statistically significant differences(P<0.05),and the pathological changes in lung tissue worsened.The results suggest that cold exposure can exacerbate LPS-induced acute lung injury,and this damaging effect may be associated with the TLR4 signaling pathway.