右美托咪定对博莱霉素诱导的肺纤维化小鼠TLR4/MyD88信号通路的影响

Impacts of dexmedetomidine on TLR4/MyD88 signaling pathway in bleomycin-induced pulmonary fibrosis mice

摘要:

目的： 探究右美托咪定（dexmedetomidine，DEX）对博莱霉素（bleomycin，BLM）诱导的肺纤维化小鼠的保护作用以及对Toll样受体4/髓样分化因子（toll-like receptor 4/myeloid differentiation factor88，TLR4/MyD88）信号通路的影响。方法： 通过注射BLM诱导实验性小鼠肺纤维化。将小鼠分为生理盐水（Sham）组、BLM组、BLM+DEX组、BLM+PFD组和BLM+DEX+LPS组。苏木素-伊红（hematoxylin and eosin，HE）染色和Masson染色分析肺组织病理学变化；免疫组织化学检测纤连蛋白（fibronectin，Fn）、α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）和Ⅰ型胶原蛋白（collagenⅠ）表达情况；原位缺口末端标记法（TdT mediated dUTP nick end labeling，TUNEL）检测肺组织中细胞凋亡；检测支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中总细胞数量及蛋白质含量；Western blot检测肺组织中TLR4/MyD88信号通路和细胞凋亡相关蛋白水平。结果： 与Sham组相比，BLM刺激后肺组织中肺泡炎评分[（2.56±0.24）分vs.（0.15±0.02）分]和Ashcroft评分[（5.68±0.52）分vs.（0.09±0.01）分]明显增高，Fn[（63.63±5.48）% vs.（25.12±2.16）%]、α-SMA[（58.63±5.03）% vs.（17.56±1.25）%]和collagen Ⅰ[（55.32±5.16）% vs.（12.03±1.20）%]表达升高（P<0.05），肺部炎症程度和纤维化程度增加；同时TUNEL阳性细胞率增高，细胞中bcl-2相关X蛋白（bcl-2 associated X protein，Bax）水平增高，B淋巴细胞瘤-2蛋白（B-cell lymphoma-2 protein，Bcl-2）水平降低（P<0.05）；BALF中总细胞数量[（3.54±0.06）×10⁵个/mL vs.（0.98±0.07）×10⁵个/mL]和蛋白质含量[（2.85±0.20）mg/mL vs.（0.29±0.03）mg/mL]升高（P<0.05）；肺组织中TLR4和MyD88蛋白水平升高（P<0.05）。DEX治疗可减轻BLM诱导的小鼠肺纤维化和炎症，降低TUNEL阳性细胞率，并逆转Fn、α-SMA、collagen Ⅰ、Bax和Bcl-2表达（P<0.05）；同时DEX也降低BLM诱导的小鼠BALF中总细胞数量和蛋白质含量（P<0.05）。此外，DEX降低BLM诱导的小鼠肺组织中TLR4和MyD88蛋白水平（P<0.05）。吡非尼酮（pirfenidone，PFD）与DEX具有相似的作用效果。脂肪酶（lipase，LPS）可以部分逆转DEX对BLM诱导的肺纤维化小鼠保护作用（P<0.05）。结论： DEX可能通过抑制TLR4/MyD88信号通路改善BLM诱导的小鼠肺纤维化。

Abstract:

Objective: To investigate the protective effect of dexmedetomidine (DEX) on bleomycin (BLM) -induced pulmonary fibrosis in mice and its effect on toll-like receptor 4/myeloid differentiation factor88 (TLR4/MyD88) signaling pathway. Methods: Experimental mouse pulmonary fibrosis was induced by injection of BLM. The mice were grouped into: saline (Sham) group, BLM group, BLM+DEX group, BLM+PFD group, and BLM+DEX+LPS group. The pathological changes of lung tissue were analyzed by hematoxylin and eosin (HE) and Masson staining; the expression of fibronectin (Fn) , α-smooth muscle actin (α-SMA) and type Ⅰ collagen (collagen Ⅰ) was measured by immunohistochemistry; the apoptosis in lung tissue was measured by TdT mediated dUTP nick end labeling (TUNEL) ; the total cell number and protein content in bronchoalveolar lavage fluid (BALF) were measured; and the TLR4/MyD88 signaling pathway and apoptosis-related protein levels in lung tissue were detected by Western blot. Results: Compared with the Sham group, the alveolitis score [(2.56±0.24) points vs. (0.15±0.02) points] and Ashcroft score [(5.68±0.52) points vs. (0.09±0.01) points] in lung tissue after BLM stimulation increased significantly, the expression of Fn [(63.63±5.48) % vs. (25.12±2.16) %], α-SMA [(58.63 ± 5.03) % vs. (17.56±1.25) %] and collagenⅠ [(55.32 ± 5.16) % vs. (12.03±1.20) %] increased (P<0.05) , and the degrees of lung inflammation and fibrosis increased. Meantime, the rate of TUNEL positive cells increased, the level of bcl-2 associated X protein (Bax) protein in the cells increased, and the level of B-cell lymphoma-2 (Bcl-2) protein decreased (P<0.05) ; the total cell number [(3.54±0.06) ×10⁵/mL vs. (0.98±0.07) ×10⁵/mL] and protein content [(2.85±0.20) mg/mL vs. (0.29±0.03) mg/mL] increased in BALF (P<0.05) ; the protein levels of TLR4 and MyD88 in lung tissue were increased (P<0.05). DEX treatment attenuated BLM-induced pulmonary fibrosis and inflammation in mice, decreased the rate of TUNEL-positive cells, and reversed the expression of Fn, α-SMA, collagen I, Bax and Bcl-2 (P<0.05) ; meanwhile, DEX also decreased the total cell number and protein content in BLM-induced mouse BALF (P<0.05) ; in addition, DEX reduced TLR4 and MyD88 protein levels in BLM-induced mouse lung tissue (P<0.05). Pirfenidone (PFD) and DEX had similar effects; lipase (LPS) was able to partially reverse the protective effect of DEX on BLM-induced pulmonary fibrosis in mice (P<0.05). Conclusion: DEX may improve BLM-induced pulmonary fibrosis in mice by inhibiting TLR4/MyD88 signaling pathway.