丹参通络解毒汤对博莱霉素诱导的肺纤维化小鼠肿瘤坏死因子样弱凋亡诱导因子/成纤维细胞生长因子诱导14通路的影响

Effect of Danshen Tongluo Jiedu decoction on tumor necrosis factor-like weak inducer of apoptosis/fibroblast growth factor-inducible 14 pathway in mice with pulmonary fibrosis induced by bleomycin

目的观察丹参通络解毒汤对博莱霉素诱导的肺纤维化小鼠肿瘤坏死因子样弱凋亡诱导因子(TWEAK)/成纤维细胞生长因子诱导14(Fn14)通路的影响,探讨其对小鼠肺纤维化的影响及机制。方法取C57B/L小鼠50只随机分为空白组、模型组、1,25-二羟维生素D_(3)[1,25(OH)_(2)D_(3)]组、丹参通络解毒汤组、丹参通络解毒汤+TWEAK/Fn14抑制剂PDL192组。模型组、1,25(OH)_(2)D_(3)组、丹参通络解毒汤组、丹参通络解毒汤+PDL192组小鼠气管内注射博莱霉素建立小鼠肺纤维化模型。造模成功后,空白组和模型组小鼠每日灌胃生理盐水10 mL·kg^(-1);1,25(OH)_(2)D_(3)组小鼠每日经腹腔注射1,25(OH)_(2)D_(3)5μg·kg^(-1);丹参通络解毒汤组小鼠每日灌胃丹参通络解毒汤汤剂0.039 mL·g^(-1)(按成人60 kg体质量计算,相当于生药15.65 g·kg^(-1));丹参通络解毒汤+PDL192组小鼠每日灌胃丹参通络解毒汤汤剂0.039 mL·g^(-1),并经腹腔注射PDL1923 mg·kg^(-1);各组小鼠均每日给药1次,连续给药28 d;末次给药后12 h测体质量,然后经腹腔注射戊巴比妥钠进行麻醉、固定,采集肺支气管肺泡灌洗液(BALF),开胸取肺组织,测肺质量,计算小鼠肺系数。采用酶标仪检测5组小鼠BALF中白细胞介素1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、白细胞介素4(IL-4)水平。苏木精-伊红染色和Masson三色法染色法观察5组小鼠肺组织病理学变化。实时荧光定量聚合酶链式反应法检测5组小鼠肺组织TWEAK、Fn14、转化生长因子β_(1)(TGF-β_(1))mRNA表达。Western blot法检测5组小鼠肺组织炎症细胞因子TNF-α、IL-1β、细胞间黏附分子(ICAM-1)、血管细胞黏附分子1(VCAM-1)及肺组织TWEAK/Fn14通路因子TWEAK、Fn14、TGF-β_(1)、α-平滑肌肌动蛋白(α-SMA)、母亲信号蛋白同源物3(Smad3)、磷酸化母亲信号蛋白同源物3(p-Smad3)、信号传导和转录激活因子3(STAT3)、酪氨酸磷酸化STAT3(p-STAT3)蛋白相对表达量。结果空白组小鼠肺组织结构正常,无结缔组织增生,无明显胶原沉积;模型组小鼠肺组织基本形态结构被破坏,支气管结构较为模糊,部分支气管上皮细胞坏死,间质周围炎症细胞浸润,肺组织有大量的胶原沉积;1,25(OH)_(2)D_(3)组小鼠肺组织基本形态结构被破坏,上皮细胞坏死,坏死区域肺泡腔较为狭窄,内含大量细胞碎片和炎症细胞,部分区域可见成纤维细胞增生,胶原沉积明显减少;丹参通络解毒汤组小鼠部分区域肺组织结构被破坏,支气管结构较为完整清晰,部分肺泡结构模糊,肺泡腔狭窄,间质及周围未见水肿或炎症细胞浸润,胶原沉积明显减少;丹参通络解毒汤+PDL192组小鼠肺组织结构较为正常,各级支气管结构完整清晰,肺泡结构较为清晰,肺泡腔内可见少量炎症细胞散在分布,间质及周围未见水肿或炎症细胞浸润,胶原沉积明显减少。空白组、模型组、1,25(OH)_(2)D_(3)组、丹参通络解毒汤组、丹参通络解毒汤+PDL192组小鼠体质量比较差异均无统计学意义(P>0.05)。与空白组比较,模型组、1,25(OH)_(2)D_(3)组、丹参通络解毒汤组小鼠肺系数显著增高(P<0.05);与模型组比较,丹参通络解毒汤组、丹参通络解毒汤+PDL192组小鼠肺系数显著降低(P<0.05);与1,25(OH)_(2)D_(3)组比较,丹参通络解毒汤+PDL192组小鼠肺系数显著降低(P<0.05);1,25(OH)_(2)D_(3)组、丹参通络解毒汤+PDL192组小鼠肺系数与丹参通络解毒汤组比较差异无统计学意义(P>0.05)。与空白组比较,模型组、1,25(OH)_(2)D_(3)组、丹参通络解毒汤组小鼠BALF中IL-1β、TNF-α、IL-4水平显著升高(P<0.05),丹参通络解毒汤+PDL192组小鼠BALF中IL-1β水平显著升高(P<0.05);与模型组比较,1,25(OH)_(2)D_(3)组、丹参通络解毒汤组、丹参通络解毒汤+PDL192组小鼠BALF中IL-1β、TNF-α、IL-4水平显著降低(P<0.05);与1,25(OH)_(2)D_(3)组比较,丹参通络解毒汤组、丹参通络解毒汤+PDL192组小鼠BALF中IL-1β、IL-4水平显著降低(P<0.05);与丹参通络解毒汤组比较,丹参通络解毒汤+PDL192组小鼠BALF中IL-1β、IL-4水平显著降低(P<0.05)。与空白组比较,模型组小鼠肺组织TWEAK、Fn14、TGF-β1 mRNA相对表达量及1,25(OH)_(2)D_(3)组小鼠肺组织TWEAK mRNA相对表达量显著升高(P<0.05);与模型组比较,丹参通络解毒汤组小鼠肺组织Fn14 mRNA相对表达量及丹参通络解毒汤+PDL192组小鼠肺组织TWEAK、Fn14、TGF-β1 mRNA相对表达量显著降低(P<0.05)。1,25(OH)_(2)D_(3)组、丹参通络解毒汤组、丹参通络解毒汤+PDL192组小鼠肺组织TWEAK、Fn14、TGF-β1 mRNA相对表达量各组间比较差异无统计学意义(P>0.05)。与空白组比较,模型组小鼠肺组织TNF-α、IL-1β、ICAM-1、VCAM-1蛋白相对表达量及1,25(OH)_(2)D_(3)组、丹参通络解毒汤组小鼠肺组织TNF-α、IL-1β蛋白相对表达量显著升高(P<0.05);与模型组比较,丹参通络解毒汤组、丹参通络解毒汤+PDL192组小鼠肺组织TNF-α、IL-1β蛋白相对表达量显著降低(P<0.05);与1,25(OH)_(2)D_(3)组比较,丹参通络解毒汤组和丹参通络解毒汤+PDL192组小鼠肺组织IL-1β蛋白相对表达量显著降低(P<0.05);与丹参通络解毒汤组比较,丹参通络解毒汤+PDL192组小鼠肺组织IL-1β蛋白相对表达量显著降低(P<0.05)。与空白组比较,模型组小鼠肺组织中TWEAK、Fn14、TGF-β1、α-SMA、p-Smad3、STAT3、p-STAT3蛋白相对表达量显著升高(P<0.05),1,25(OH)_(2)D_(3)组小鼠肺组织中TWEAK、TGF-β1、α-SMA、p-Smad3、STAT3、p-STAT3蛋白相对表达量显著升高(P<0.05),丹参通络解毒汤组、丹参通络解毒汤+PDL192组小鼠肺组织中TWEAK、α-SMA、p-Smad3、STAT3蛋白相对表达量显著升高(P<0.05);与模型组比较,1,25(OH)_(2)D_(3)组小鼠肺组织p-Smad3蛋白相对表达量显著降低(P<0.05),丹参通络解毒汤组、丹参通络解毒汤+PDL192组小鼠肺组织TWEAK、Fn14、TGF-β1、α-SMA、p-Smad3、STAT3蛋白相对表达量显著降低(P<0.05)。丹参通络解毒汤组、丹参通络解毒汤+PDL192组小鼠肺组织Fn14、TGF-β1、α-SMA、Smad3、p-Smad3、p-STAT3蛋白相对表达量及丹参通络解毒汤小鼠肺组织TWEAK、STAT3蛋白相对表达量与1,25(OH)_(2)D_(3)组比较差异无统计学意义(P>0.05);丹参通络解毒汤+PDL192组小鼠肺组织中TWEAK、STAT3蛋白相对表达量与1,25(OH)_(2)D_(3)组比较显著降低(P<0.05)。结论丹参通络解毒汤能够减轻博莱霉素诱导的小鼠肺纤维化,其机制可能与抑制肺组织炎症和TWEAK/Fn14信号通路有关。

Objective To observe the effects of Danshen Tongluo Jiedu decoction on tumor necrosis factor-like weak inducer of apoptosis(TWEAK)/fibroblast growth factor-inducible 14(Fn14)pathway in mice with pulmonary fibrosis,and to investigate its effects and mechanism on pulmonary fibrosis in mice.Methods A total of fifty C57B/L mice were randomly divided into blank group,model group,positive control group{1,25-dihydroxyvitamin D_(3)[1,25(OH)_(2)D_(3)]group},Danshen Tongluo Jiedu decoction group and Danshen Tongluo Jiedu decoction+TWEAK/Fn14 inhibitor(PDL192)group.The mice in the model group,1,25(OH)_(2)D_(3) group,Danshen Tongluo Jiedu decoction group and Danshen Tongluo Jiedu decoction+PDL192 group were injected intratracheally with bleomycin to establish pulmonary fibrosis model.After successful modeling,the mice in the control group and model group were gavaged with saline 10 mL·kg^(-1) daily;the mice in the 1,25(OH)_(2)D_(3) group were injected with 5μg·kg^(-1) of 1,25(OH)_(2)D_(3) intraperitoneally daily;the mice in the Danshen Tongluo Jiedu decoction group were gavaged with Danshen Tongluo Jiedu decoction 0.039 mL·g^(-1) daily(equivalent to 15.65 g·kg^(-1) of raw drug based on adult body mass of 60 kg);the mice in the Danshen Tongluo Jiedu decoction+PDL192 group were gavaged with Danshen Tongluo Jiedu decoction 0.039 mL·g^(-1) and injected with PDL1923 mg·kg^(-1) intraperitoneally;the mice in each group were administered once a day for 28 d.The body mass of mice in each group was measured at 12 h after the last dose,then the mice were anesthetized and fixed by intraperitoneal injection of sodium pentobarbital,bronchoalveolar lavage fluid(BALF)was collected,lung tissue was taken from the open chest,lung mass was measured,and the lung coefficients of mice were calculated.The levels of interleukin 1β(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin 4(IL-4)in BALF of mice in the five groups were measured by enzyme marker.The histopathological changes of the lungs of mice in the five groups were observed by hematoxylin-eosin(HE)staining and Masson′s trichrome staining.The mRNA expression levels of TWEAK,Fn14 and transforming growth factorβ_(1)(TGF-β_(1))were detected by real-time fluorescence quantitative polymerase chain reaction.The relative expression levels of inflammatory cytokines TNF-α,IL-1β,intercellular adhesion molecule(ICAM-1),vascular cell adhesion molecule 1(VCAM-1),lung tissue TWEAK/Fn14 pathway factors TWEAK,Fn14,TGF-β_(1),α-smooth muscle actin(α-SMA),mothers against decapentaplegic homolog 3(Smad3),phosphoSMAD3(p-Smad3),signal transducer and activator of transcription 3(STAT3),tyrosine phosphorylated STAT3(p-STAT3)in lung tissue of mice in the five groups were detected by Western blot.Results The lung tissue structure of the mice in the blank group was normal,without connective tissue proliferation and obvious collagen deposition;the basic morphology and structure of the lung tissue in the model group were destroyed,the bronchial structure was blurred,some bronchial epithelial cells were necrotic,inflammatory cells around the interstitium were infiltrated,and there was a large amount of collagen deposition in the lung tissue;the basic morphological structure of lung tissue was destroyed of mice in the 1,25(OH)_(2)D_(3) group,with necrosis of epithelial cells,narrower alveolar lumen in the necrotic area,containing a large number of cellular debris and inflammatory cells,with fibroblast proliferation and significantly reduced collagen deposition visible in some areas;in the Danshen Tongluo Jiedu decoction group,some areas of the lung tissue structure were destroyed,the bronchial structure was relatively complete and clear,some alveolar structures were blurred,the alveolar cavity was narrow,there was no edema or inflammatory cell infiltration in the interstitium and surrounding areas,and collagen deposition was significantly reduced;the lung tissue structure of mice in the group of Danshen Tongluo Jiedu decoction+PDL192 was more normal,the bronchial structure at all levels was intact and clear,the alveolar structure was clear,a few inflammatory cells were scattered in the alveolar cavity,edema or inflammatory cell infiltration was not seen in the interstitium and surrounding area,and collagen deposition was significantly reduced.There was no significant difference in the body mass of mice among the blank group,model group,1,25(OH)_(2)D_(3) group,Danshen Tongluo Jiedu decoction group and Danshen Tongluo Jiedu decoction+PDL192 group(P>0.05).The lung coefficient of mice in the model group,the 1,25(OH)_(2)D_(3) group and the Danshen Tongluo Jiedu decoction group was significantly higher than that in the blank group(P<0.05);the lung coefficient of mice in the Danshen Tongluo Jiedu decoction group and the Danshen Tongluo Jiedu decoction+PDL192 group was significantly lower than that in the model group(P<0.05);the lung coefficient of mice in the Danshen Tongluo Jiedu decoction+PDL192 group was significantly lower than that in the 1,25(OH)_(2)D_(3) group(P<0.05).There was no significant difference in lung coefficient of mice between 1,25(OH)_(2)D_(3) group,Danshen Tongluo Jiedu decoction+PDL192 group and Danshen Tongluo Jiedu decoction group(P>0.05).The level of IL-1β,TNF-αand IL-4 in the BALF of mice in the model group,1,25(OH)_(2)D_(3) group and Danshen Tongluo Jiedu decoction group were significantly higher than those in the blank group(P<0.05),and the level of IL-1βin the BALF of mice in the Danshen Tongluo Jiedu decoction+PDL192 group was significantly higher than that in the blank group(P<0.05).The levels of IL-1β,TNF-αand IL-4 in BALF of mice in the 1,25(OH)_(2)D_(3) group,Danshen Tongluo Jiedu decoction group and Danshen Tongluo Jiedu decoction+PDL192 group were significantly lower than those in the model group(P<0.05).The levels of IL-1βand IL-4 in the BALF of mice in the Danshen Tongluo Jiedu decoction group and Danshen Tongluo Jiedu decoction+PDL192 group were significantly lower than those in the 1,25(OH)_(2)D_(3) group(P<0.05).The levels of IL-1βand IL-4 in BALF of mice in the Danshen Tongluo Jiedu decoction+PDL192 group were significantly lower than those in the Danshen Tongluo Jiedu decoction group(P<0.05).The relative expression levels of TWEAK,Fn14 and TGF-β_(1) mRNA in lung tissues of mice in the model group and the relative expression level of TWEAK mRNA in lung tissues of mice in the 1,25(OH)_(2)D_(3) group were significantly higher than those in the blank group(P<0.05).The relative expression level of Fn14 mRNA in the lung tissue of mice in the Danshen Tongluo Jiedu decoction group and the expression levels of TWEAK,Fn14 and TGF-β_(1) mRNA in the lung tissue of mice in the Danshen Tongluo Jiedu decoction+PDL192 group were significantly lower than those in the model group(P<0.05).There was no significant difference in the relative expression of TWEAK,Fn14,TGF-β_(1) mRNA in the lung tissue of mice among the 1,25(OH)_(2)D_(3) group,Danshen Tongluo Jiedu decoction group,Danshen Tongluo Jiedu decoction+PDL192 group(P>0.05).The relative expression levels of TNF-α,IL-1β,ICAM-1 and VCAM-1 protein in the lung tissue of mice in the model group and the relative expression levels of TNF-αand IL-1βprotein in the lung tissue of mice in the 1,25(OH)_(2)D_(3) group and the Danshen Tongluo Jiedu decoction group were significantly higher than those in the blank group(P<0.05);the relative expression levels of TNF-αand IL-1βin lung tissues of mice in the Danshen Tongluo Jiedu decoction and Danshen Tongluo Jiedu decoction+PDL192 groups were significantly lower than those in the model group(P<0.05).The relative expression level of IL-1βprotein in lung tissue of mice in the Danshen Tongluo Jiedu decoction group and Danshen Tongluo Jiedu decoction+PDL192 group was significantly lower than that in the 1,25(OH)_(2)D_(3) group(P<0.05).The relative expression level of IL-1βprotein in lung tissue of mice in the Danshen Tongluo Jiedu decoction+PDL192 group was significantly lower than that in the Danshen Tongluo Jiedu decoction group(P<0.05).The relative expression levels of TWEAK,Fn14,TGF-β_(1),α-SMA,p-Smad3,STAT3 and p-STAT3 protein in lung tissue of mice in the model group were significantly higher than those in the blank group(P<0.05),and the relative expression levels of TWEAK,TGF-β_(1),α-SMA,p-Smad3,p-STAT3 and p-STAT3 protein in lung tissue of mice in the 1,25(OH)_(2)D_(3) group were significantly higher than those in the blank group(P<0.05),and the relative expression levels of TWEAK,α-SMA,p-Smad3,STAT3 protein were significantly higher than those in the blank group(P<0.05).The relative expression level of p-Smad3 protein in the lung tissue of mice in the 1,25(OH)_(2)D_(3) group was significantly lower than that in the model group(P<0.05),and the relative expression levels of TWEAK,Fn14,TGF-β_(1),α-SMA,p-Smad3 and STAT3 protein in the lung tissue of mice in the Danshen Tongluo Jiedu decoction group and Danshen Tongluo Jiedu decoction+PDL192 group were significantly lower than those in the model group(P<0.05).The relative expressions of Fn14,TGF-β_(1),α-SMA,Smad3,p-Smad3 and p-STAT3 in lung tissue of mice in the Danshen Tongluo Jiedu decoction group,Danshen Tongluo Jiedu decoction+PDL192 group and the relative expressions of TWEAK and STAT3 in lung tissue of mice in the Danshen Tongluo Jiedu decoction group had no significant difference compared with those in the 1,25(OH)_(2)D_(3) group(P>0.05).The relative expression levels of TWEAK and STAT3 protein in lung tissue of mice in the Danshen Tongluo Jiedu decoction+PDL192 group were significantly lower than those in the 1,25(OH)_(2)D_(3) group(P<0.05).Conclusion Danshen Tongluo Jiedu decoction can reduce bleomycin-induced pulmonary fibrosis in mice,and the mechanism may be related to the inhibition of lung tissue inflammation and TWEAK/Fn14 signaling pathway.