海风藤组方对慢性硬膜下血肿大鼠炎性介质滴度表达及其依赖性新生血管的影响

Effect of Formulae Composed of Caulis Piperis Kadsurae and Other Medicinals on the Expression of Inflammatory Mediator Titers and Its Dependent Neovascularization in Rats with Chronic Subdural Hematoma

目的:观察海风藤组方对慢性硬膜下血肿大鼠炎性反应介质单核细胞趋化蛋白-1(MCP-1)和环氧化酶-2(COX-2)及其依赖性新生血管的影响,探讨海风藤组方对慢性硬膜下血肿大鼠的治疗作用及机制。方法:SD大鼠60只,采用自体血注射法建立大鼠慢性硬膜下血肿模型,共造模成功48只,按照随机数字表法将大鼠分为假手术组10只、模型组9只、阳性对照组9只、海风藤组方低剂量组10只、海风藤组方高剂量组10只。假手术组和模型组灌胃蒸馏水,阳性对照组灌胃阿托伐他汀钙片水溶液,海风藤组方低剂量组、高剂量组灌胃海风藤组方药液,每日1次,连续灌胃14 d后取材进行检验分析。观察大鼠体质量及大鼠神经行为评分;MRI扫描观察血肿体积;酶联免疫吸附法(ELISA)检测血清和血肿液中MCP-1和COX-2水平;透射电镜观察血肿外膜结构变化;免疫组化染色法检测血肿外膜新生微血管密度;免疫印迹法检测血肿外膜组织中血管内皮生长因子(VEGF)和转化生长因子β1(TGF-β1)表达水平。结果:与假手术组相比,模型组大鼠血清MCP-1、COX-2水平升高(P<0.01),与模型组相比,阳性对照组、海风藤组方低剂量组、海风藤组方高剂量组大鼠血肿体积缩小,神经功能改善,血清及血肿液MCP-1、COX-2水平降低(P<0.01),血肿外组织VEGF、TGF-β1表达水平降低(P<0.01),血肿外膜组织新生血管密度降低(P<0.01)。与海风藤组方低剂量组相比,海风藤组方高剂量组大鼠血肿体积缩小,神经功能改善,血清及血肿液MCP-1、COX-2水平降低(P<0.05),血肿外组织VEGF、TGF-β1表达水平降低(P<0.05),血肿外膜组织新生血管密度降低(P<0.05)。结论:海风藤组方可改善大鼠神经功能,促进血肿吸收,其机制可能与抑制MCP-1、COX-2相关炎症反应及减少血管新生有关。

Objective:To observe the effects of formulae composed of Haifengteng(caulis piperis kadsurae,CPK)and other medicinals on inflammatory mediators monocyte chemoattractant protein-1(MCP-1),cyclooxygenase-2(COX-2),and neovascularization in rats with chronic subdural hematoma,and to explore the treatment action and mechanism of formulae with CPK on chronic subdural hematoma in rats.Methods:Sixty SD rats were collected and the models of chronic subbural hematoma were established by autologous blood injection.The rats were divided according to a random number table into a sham surgery group(n=10),a model group(n=9),a positive control group(n=9),a low-dose group of CPK formula(n=10),and a high-dose group of CPK formula(n=10).The sham operation group and model group were given saline by gavage,the positive control group was administrated with atorvastatin calcium tablet aqueous solution by gavage,and the low-dose and high-dose groups of CPK formula were gavaged with CPK formula decoction,once a day,for 14 consecutive days.Samples were taken from the rats for analysis after the 14 days.The body weight and neurological behavior score of the rats were observed,the hematoma volume was measured by MRI,and the levels of MCP-1 and COX-2 in serum and hematoma fluid were detected by enzyme-linked immunosorbent assay(ELISA).The changes in the structure of the hematoma outer membrane were observed by transmission electron microscopy,and the density of newly formed microvessels in the hematoma outer membrane was detected by immunohistochemical staining.The expression levels of vascular endothelial growth factor(VEGF)and transforming growth factorβ1(TGF-β1)in the hematoma outer membrane tissue were detected by Western blotting.Results:Compared with the sham group,the serum levels of MCP-1 and COX-2 in the model group were elevated(P<0.01).Compared with the model group,the hematoma volume was reduced,neurological function was improved,serum and hematoma levels of MCP-1 and COX-2 were decreased(P<0.01),the expression levels of VEGF and TGF-β1 in the hematoma external tissue(P<0.01),and the density of newly formed blood vessels in the hematoma external membrane tissue were reduced(P<0.01)in positive control group,low-dose,and high-dose groups of CPK formula.Compared with the low-dose group of CPK formula,the high-dose group of CKP formula had reduced hematoma volume,improved neurological function,and decreased serum and hematoma fluid levels of MCP-1 and COX-2(P<0.05),the expression levels of VEGF and TGF-β1 in the hematoma external tissue and the density of newly formed blood vessels in the hematoma external membrane tissue were reduced(P<0.05).Conclusion:CPK formula can improve the neurological function of rats and promote hematoma absorption.Its mechanism may be related to the inhibition of MCP-1 and COX-2 related inflammatory reactions and the reduction of angiogenesis.