大麻素2型受体激动剂JWH133干预对肺纤维化小鼠的保护作用

Protective effect of intervention with cannabinoid type-2 receptor agonist JWH133 on pulmonary fibrosis in mice

目的探讨大麻素2型受体激动剂JWH133对肺纤维化小鼠的保护作用。方法选24只C57BL/6J雄性小鼠按随机数字法分为对照组、模型组、JWH133干预组、JWH133+大麻素2型受体拮抗剂AM630拮抗组,每组6只。气管滴注博来霉素(5 mg/kg)制作小鼠肺纤维化模型,造模后第1天起,对照组小鼠腹腔注射0.9%氯化钠溶液0.1 ml,模型组小鼠腹腔注射0.9%氯化钠溶液0.1 ml,JWH133干预组小鼠腹腔注射JWH133(2.5 mg/kg,溶于生理盐水中)0.1 ml,JWH133+AM630拮抗组小鼠腹腔注射JWH133(2.5 mg/kg)和AM630(2.5 mg/kg)0.1 ml,28 d后处死所有小鼠,取肺组织,病理观察肺组织改变,并进行肺泡炎评分和Ashcroft评分,采用免疫组化测4组小鼠肺组织Ⅰ型胶原含量,酶联免疫吸附测定(ELISA)测4组小鼠血清中白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)水平,测4组小鼠肺组织中羟脯氨酸含量,Western-blot测4组小鼠肺组织中Ⅲ型胶原、α-平滑肌肌动蛋白(α-SMA)、细胞外信号调节激酶(ERK1/2)、磷酸化ERK1/2(P-ERK1/2)、磷酸化核糖体S6激酶1(P-p90RSK)蛋白表达水平;实时荧光定量聚合酶链式反应测4组小鼠肺组织中Ⅰ型胶原、Ⅲ型胶原、α-SMA mRNA表达水平。结果与对照组比,模型组小鼠肺组织病理改变加重,肺泡炎评分升高[(3.833±0.408)分比(0.833±0.408)分,P<0.05],Ashcroft评分升高[(7.333±0.516)分比(2.000±0.633)分,P<0.05],Ⅰ型胶原吸光度值升高(0.065±0.008比0.018±0.006,P<0.05),炎性细胞浸润加重,羟脯氨酸含量升高[(1.551±0.051)μg/mg比(0.974±0.060)μg/mg,P<0.05];与模型组比,JWH133干预组小鼠肺组织病理改变减轻,肺泡炎评分降低[(1.833±0.408)分,P<0.05],Ashcroft评分降低[(4.167±0.753)分,P<0.05],Ⅰ型胶原吸光度值降低(0.032±0.004,P<0.05),炎性细胞浸润减轻,羟脯氨酸含量下降[(1.148±0.055)μg/mg,P<0.05];与JWH133干预组比,JWH133+AM630拮抗组小鼠肺组织病理改变加重,肺泡炎评分和Ashcroft评分升高,Ⅰ型胶原吸光度值升高,炎性细胞浸润加重,羟脯氨酸含量升高。与对照组比,模型组小鼠肺组织α-SMA、Ⅲ型胶原、P-ERK1/2、P-p90RSK蛋白表达升高,Ⅰ型胶原、Ⅲ型胶原、α-SMA mRNA表达升高;与模型组比,JWH133干预组α-SMA(相对表达量0.60±0.17比1.34±0.19,P<0.05)、Ⅲ型胶原(相对表达量0.52±0.09比1.35±0.14,P<0.05)、P-ERK1/2(相对表达量0.32±0.11比1.14±0.14,P<0.05)、P-p90RSK(相对表达量0.43±0.14比1.15±0.07,P<0.05)蛋白表达下降;Ⅰ型胶原(相对表达量2.190±0.362比5.078±0.792,P<0.05)、Ⅲ型胶原(相对表达量1.750±0.290比4.935±0.456,P<0.05)、α-SMA(相对表达量1.588±0.060比5.192±0.506,P<0.05)mRNA表达降低;与JWH133干预组比,JWH133+AM630拮抗组小鼠肺组织α-SMA、Ⅲ型胶原、P-ERK1/2、P-p90RSK蛋白表达升高,Ⅲ型胶原、α-SMA mRNA表达升高。结论大麻素2型受体激动剂JWH133可抑制小鼠肺组织炎症,通过改善细胞外基质沉积减轻肺纤维化,其机制可能与抑制ERK1/2-RSK1信号通路有关。

Objective JWH133,a cannabinoid type 2 receptor agonist,was tested for its ability to protect mice from bleomycin-induced pulmonary fibrosis.Methods By using a random number generator,24 C57BL/6J male mice were randomly divided into the control group,model group,JWH133 intervention group,and JWH133+a cannabinoid type-2 receptor antagonist(AM630)inhibitor group,with 6 mice in each group.A mouse pulmonary fibrosis model was established by tracheal instillation of bleomycin(5 mg/kg).Starting from the first day after modeling,the control group mice were intraperitoneally injected with 0.1 ml of 0.9%sodium chloride solution,and the model group mice were intraperitoneally injected with 0.1 ml of 0.9%sodium chloride solution.The JWH133 intervention group mice were intraperitoneally injected with 0.1 ml of JWH133(2.5 mg/kg,dissolved in physiological saline),and the JWH133+AM630 antagonistic group mice were intraperitoneally injected with 0.1 ml of JWH133(2.5 mg/kg)and AM630(2.5 mg/kg).After 28 days,all mice were killed;the lung tissue was obtained,pathological changes were observed,and alveolar inflammation scores and Ashcroft scores were calculated.The content of typeⅠcollagen in the lung tissue of the four groups of mice was measured using immunohistochemistry.The levels of interleukin 6(IL-6)and tumor necrosis factorα(TNF-α)in the serum of the four groups of mice were measured using enzyme-linked immunosorbent assay(ELISA),and the content of hydroxyproline(HYP)in the lung tissue of the four groups of mice was measured.Western blotting was used to measure the protein expression levels of typeⅢcollagen,α-smooth muscle actin(α-SMA),extracellular signal regulated kinase(ERK1/2),phosphorylated P-ERK1/2(P-ERK1/2),and phosphorylated ribosome S6 kinase type 1(P-p90RSK)in the lung tissue of mice in the four groups.Real-time quantitative polymerase chain reaction was used to measure the expression levels of collagenⅠ,collagenⅢ,andα-SMA mRNA in the lung tissue of the four groups of mice.Results Compared with the control group,the pathological changes in the lung tissue of the model group mice worsened,with an increase in alveolar inflammation score(3.833±0.408 vs.0.833±0.408,P<0.05),an increase in Ashcroft score(7.333±0.516 vs.2.000±0.633,P<0.05),an increase in typeⅠcollagen absorbance value(0.065±0.008 vs.0.018±0.006,P<0.05),an increase in inflammatory cell infiltration,and an increase in hydroxyproline levels[(1.551±0.051)μg/mg vs.(0.974±0.060)μg/mg,P<0.05].Compared with the model group,the JWH133 intervention group showed reduced pathological changes in lung tissue,decreased alveolar inflammation score(1.833±0.408,P<0.05),decreased Ashcroft score(4.167±0.753,P<0.05),decreased typeⅠcollagen absorbance value(0.032±0.004,P<0.05),reduced inflammatory cell infiltration,and decreased hydroxyproline levels[(1.148±0.055)μg/mg,P<0.05].Compared with the JWH133 intervention group,the JWH133+AM630 antagonistic group showed more severe pathological changes in the lung tissue of mice,increased alveolar inflammation score and Ashcroft score,increased typeⅠcollagen absorbance value,increased inflammatory cell infiltration,and increased hydroxyproline levels.Compared with the control group,the expression ofα-SMA,typeⅢcollagen,P-ERK1/2,and P-p90RSK proteins in the lung tissue of the model group mice increased,while the expression of typeⅠcollagen,typeⅢcollagen,andα-SMA mRNA increased.Compared with the model group,the protein expression ofα-SMA(relative expression 0.60±0.17 vs.1.34±0.19,P<0.05),typeⅢcollagen(relative expression 0.52±0.09 vs.1.35±0.14,P<0.05),P-ERK1/2(relative expression 0.32±0.11 vs.1.14±0.14,P<0.05),and P-p90RSK(relative expression 0.43±0.14 vs.1.15±0.07,P<0.05)decreased in the JWH133 intervention group.The typeⅠcollagen mRNA(2.190±0.362 vs.5.078±0.792,P<0.05),typeⅢcollagen mRNA(1.750±0.290 vs.4.935±0.456,P<0.05),andα-SMA mRNA(1.588±0.060 vs.5.192±0.506,P<0.05)decreased.Compared with the JWH133 intervention group,the JWH133+AM630 antagonistic group increased the expression ofα-SMA,typeⅢcollagen,P-ERK1/2,and P-p90RSK protein in the lung tissue of mice,and increased the expression of typeⅢcollagen andα-SMA mRNA.Conclusion In mice with bleomycin-induced pulmonary fibrosis,the cannabinoid type-2 receptor agonist JWH133 inhibited inflammation and improved extracellular matrix deposition,which alleviated lung fibrosis.The underlying mechanism of action may be related to the activation of the ERK1/2-RSK1 signaling pathway.