金蝉花多糖改善矽肺小鼠肺纤维化的作用研究

Research on ameliorating pulmonary fibrosis in silicosis mice of Cordyceps cicadae polysaccharides

目的通过气管滴注二氧化硅(SiO 2)颗粒构建小鼠矽肺模型,探究金蝉花多糖(CCP)对矽肺小鼠肺纤维化的改善作用和机制。方法于2023年5月,提取分离得到CCP,采用高效液相色谱和傅里叶变换红外光谱分析其单糖组成和官能团构成。C57BL/6J小鼠气管滴注50μl 50 mg/ml的SiO 2混悬液以制备矽肺小鼠模型,然后将造模成功的小鼠随机分为模型组、CCP干预组[低剂量组(LCCP组)、中剂量组(MCCP组)和高剂量组(HCCP组)],对照组滴注50μl生理盐水,每组8只。CCP干预组每天分别灌胃一次100、200和400 mg/kg的CCP溶液,对照组和模型组灌胃生理盐水,连续给药30 d。记录小鼠体重并计算肺系数,采用HE和Masson染色检测小鼠肺组织的病理形态学变化;ELISA法测定肺组织中纤维化相关指标[羟脯胺酸(HYP)、结缔组织生长因子(CTGF)和基质金属蛋白酶-2(MMP-2)]、肺组织和肺泡灌洗液中促炎因子[肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和白细胞介素6(IL-6)]的含量;免疫组化法测定小鼠肺组织Ⅰ型胶原蛋白(CollagenⅠ)的表达;Western blot法检测肺组织中转化生长因子β1(TGF-β1)、P-Smad2、α-平滑肌肌动蛋白(α-SMA)、Toll样受体4(TLR4)、核因子κB p65亚基(NF-κBp65)和髓性分化原发应答基因88(MyD88)的相对表达量。结果CCP总糖含量为86.78%,由D-甘露糖、D-鼠李糖、D-葡萄糖和D-半乳糖组成,其摩尔比为12.71∶1.53∶1.00∶12.64,红外光谱表明具有多糖的特征基团。与对照组比较,模型组小鼠体重降低,肺系数升高,肺组织中HYP、CTGF和MMP-2的含量升高,肺组织和肺泡灌洗液中TNF-α、IL-1β和IL-6含量升高(P<0.05);肺组织显示大量炎症细胞浸润和胶原纤维沉积,矽肺纤维化程度严重;肺组织CollagenⅠ表达上调,TGF-β1、P-Smad2/Smad2、α-SMA、TLR4、NF-κBp65和MyD88的相对蛋白表达水平升高(P<0.05)。与模型组比较,MCCP组和HCCP组小鼠体重升高,肺系数降低,肺组织中HYP、CTGF和MMP-2的含量降低,肺组织和肺泡灌洗液中TNF-α、IL-1β和IL-6含量降低(P<0.05);肺部炎症细胞浸润减轻,纤维化程度具有不同程度的改善;肺组织CollagenⅠ表达下调,TGF-β1、P-Smad2/Smad2、α-SMA、TLR4、NF-κBp65和MyD88的相对蛋白表达水平降低(P<0.05)。结论CCP可通过抑制TGF-β1/Smad通路和TLR4/核因子κB(NF-κB)通路的激活,降低肺组织中纤维化相关指标和促炎因子的水平,改善SiO 2颗粒导致的小鼠肺部炎症和矽肺纤维化。

Objective A mouse silicosis model was constructed by injecting silicon dioxide(SiO 2)particles into the trachea to explore the effect and mechanism of Cordyceps cicadae polysaccharides(CCP)on ameliorating pulmonary fibrosis in silicosis mice.Methods In May 2023,CCP were extracted and isolated,the monosaccharide composition and functional group composition were analyzed by high performance liquid chromatography and Fourier transform infrared spectroscopy.C57BL/6J mice were injected with 50μl 50 mg/ml SiO 2 suspension to construct silicosis mouse model,which were then randomly divided into model group,CCP intervention groups[low dose group(LCCP group),medium dose group(MCCP group)and high dose group(HCCP group)],the control group was administered by physiological saline,8 mice in each group.Mice in the CCP intervention groups received oral gavage administration once daily with CCP solution(100,200 and 400 mg/kg),while control group and model group received physiological saline,lasted for 30 days.The body weight of mice was recorded and the lung coefficient was calculated.The pathomorphological changes of mouse lung tissue were determined by HE and Masson staining.The contents of fibrosis indexes[hydroxyproline acid(HYP),connective tissue growth factor(CTGF)and matrix metallopeptidase 2(MMP-2)]of lung tissue and the pro-inflammatory factors[tumor necrosis factor-alpha(TNF-α),interleukin-1 beta(IL-1β)and interleukin-6(IL-6)]of lung tissue and alveolar lavage fluid were determined by ELISA.The expression level of CollagenⅠwas determined by immunohistochemistry.The relative protein expression levels of transforming growth factor-β1(TGF-β1),P-Smad2,α-smooth muscle actin(α-SMA),Toll-like receptor 4(TLR4),nuclear factor kappa-B p65(NF-κBp65)and myeloid differentiation primary response gene 88(MyD88)in lung tissue were determined by Western blot.Results The total sugar content of the CCP was 86.78%,composed of D-mannose,D-rhamnose,D-glucose and D-galactose,with a molar ratio of 12.71∶1.53∶1.00∶12.64.The infrared spectrum indicated the characteristic groups of its polysaccharides.Compared with the control group,the body weight of mice in the model group was decreased,lung coefficient was increased,the contents of HYP,CTGF and MMP-2 in lung tissue were increased,and the contents of TNF-α,IL-1βand IL-6 in lung tissue and alveolar lavage fluid were increased(P<0.05).The mice lung showed massive inflammatory cell infiltration and collagen fiber deposition,and the silicosis fibrosis was severe.The expression of CollagenⅠin lung tissue of model group was increased,and the proteins expression levels of TGF-β1,P-Smad2/Smad2,α-SMA,TLR4,NF-κBp65 and MyD88 were increased in mouse lung tissue(P<0.05).Compared with the model group,the body weights of mice in the MCCP and HCCP groups were increased,the lung coefficients were decreased,the contents of HYP,CTGF and MMP-2 in lung tissue were decreased,and the contents of TNF-α,IL-1βand IL-6 in lung tissue and alveolar lavage fluid were decreased(P<0.05).The inflammatory cell infiltration in the lung was reduced,and the degree of fibrosis was improved to varying degrees.The expression level of CollagenⅠwas down-regulated in the lung tissue of MCCP and HCCP groups,and the protein expression levels of TGF-β1,P-Smad2/Smad2,α-SMA,TLR4,NF-κBp65 and MyD88 were decreased in lung tissue(P<0.05).Conclusion The CCP could reduce the levels of fibrosis-related indicators and pro-inflammatory factors in lung tissue,ameliorating mouse lung inflammation and silicosis fibrosis caused by SiO 2 particles by inhibiting the activation of TGF-β1/Smad pathway and TLR4/nuclear factor kappa-B(NF-κB)pathway.