活血化瘀法对慢性盆腔炎大鼠炎症细胞因子及粘连相关免疫分子影响的研究
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摘要
目的:通过观察慢性盆腔炎模型大鼠血清、子宫组织中炎症细胞因子及粘连相关免疫分子的变化,探讨活血化瘀法治疗慢性盆腔炎(CPID)的免疫调节机制。
方法:以Wistar雌性大鼠为实验对象,采用混合细菌加机械损伤法建立慢性盆腔炎动物模型。将96只大鼠随机分为正常组、假手术组、模型组、桂枝茯苓胶囊高、中、低剂量组、少腹逐瘀胶囊组和金刚藤胶囊组。药物干预后,酶联免疫吸附测定法(ELISA)检测各组大鼠血清中的前炎症细胞因子和抗炎症细胞因子,前炎症细胞因子包括白介素-1β(IL-1β)、白介素-8(IL-8)、肿瘤坏死因子-α(TNF-α),抗炎症细胞因子包括白介素-4(IL-4)、白介素-10(IL-10);大鼠子宫形态学观察后,免疫组织化学法检测各组大鼠子宫组织肿瘤坏死因子-α(TNF-α)、转化生长因子-β1(TGF-β1)、血管内皮生长因子(VEGF)、细胞间粘附分子-1(ICAM-1)、基质金属蛋白酶-2(MMP-2)的表达。
结果:慢性盆腔炎模型大鼠血清前炎症细胞因子IL-1β、IL-8、TNF-α水平较正常组升高,抗炎症细胞因子IL-4、IL-10水平较正常组降低,子宫组织中的粘连相关免疫分子TNF-α、TGF-β1、VEGF、ICAM-1、MMP-2表达均较正常组升高,差异有极显著性(P<0.01)。药物干预后,各用药组大鼠血清前炎症细胞因子水平均下降,抗炎症细胞因子水平均升高,子宫组织中的MMP-2表达较模型组升高,TNF-α、TGF-β1、VEGF、ICAM-1的表达均较模型组降低(P<0.05)。桂枝茯苓中剂量组对血清和组织中的TNF-α、TGF-β1及组织中ICAM-1、MMP-2的调节作用优于少腹逐瘀组和金刚藤组(P<0.05)。桂枝茯苓胶囊干预的盆腔炎模型大鼠的炎症细胞因子和粘连相关免疫分子之间存在着显著正负相关性。
结论:活血化瘀法、清热解毒法可通过对大鼠血清、子宫组织的炎症细胞因子和粘连相关免疫分子的调节,有效消除盆腔炎模型大鼠盆腔炎症,缓解盆腔粘连,改善机体免疫功能。活血化瘀法对粘连相关免疫分子表达的调节作用明显优于清热解毒法,其在改善慢性盆腔炎症粘连的免疫调节方面表现出优势。
Objective: To explore the immunoregulation mechanism of the treatment of activating blood to remove blood stasis on chronic pelvic inflammatory disease(CPID) by observing the level of inflammatory cytokines and adhesion related molecules in serum and uterine tissue of CPID model rats.
Method:Female Wistar rats were used and the CPID model was established with mixed bacteria injection combined with mechanic injury. Ninty-six rats were randomly divided into normal group, sham operation group, model group, Guizhi Fuling high, medium and low dose group, Shaofuzhuyu group and Jingangteng group. The histomorphological changes of the rats' uteri were observed using light microscope.The enzyme-linked immunosorbent assay (ELISA) was applied to measure the concentration of pro-inflammatory cytokines (IL-1β, IL-8, TNF-α) and anti-inflammatory cytokines(IL-4, IL-10)in serum of rats in each group; the immunohistochemistry method was used to detect the expression of the following indices in rats’uterine tissue: tumor necrosis factor-α(TNF-α), transforming growth factor-β1(TGF-β1), vascular endothelial growth factor(VEGF), intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase protein -2(MMP-2).
Results:The serous levels of pro-inflammatory cytokines (IL-1β, IL-8, TNF-α)in model group were significantly higher than those in normal group; the serous levels of anti-inflammatory cytokines (IL-4, IL-10) in model group were significantly lower than those in normal group. The expression of adhesion related immune molecules(TNF-α, TGF-β1, VEGF, ICAM-1, MMP-2) in model group was higher than normal group significantly(P<0.01).After treatment, the pro-inflammatory cytokine levels were decreased while the anti-inflammatory cytokine levels were elevated,the expression of TNF-α、TGF-β1、VEGF and ICAM-1 decreased, and MMP-2 elevated(P<0.05). Guizhi Fuling medium dose group showed abetter therapeutic effects on regulating TNF-α、TGF-β1、ICAM-1、MMP-2 than Shaofuzhuyu group and Jingangteng group(P<0.05). There were positive or negative correlation between Inflammatory cytokines and adhesion related immune molecules in Guizhi Fuling medium dose group.
Conclusions: The method of activating blood to remove blood stasis and clearing away heat and toxin could adjust the level of inflammatory cytokines and various immune-related adhesion molecule levels, thus adjust the immune balance effectively so as to inhibit inflammation, prevent and alleviate the pelvic adhesions. The method of activating blood to remove blood stasis is superior to the method of clearing away heat and toxin in the regulation of adhesion related immune molecules so it has an advantage of alleviating the adhesion in CPID through certain immune mechanisms.
方法:以Wistar雌性大鼠为实验对象,采用混合细菌加机械损伤法建立慢性盆腔炎动物模型。将96只大鼠随机分为正常组、假手术组、模型组、桂枝茯苓胶囊高、中、低剂量组、少腹逐瘀胶囊组和金刚藤胶囊组。药物干预后,酶联免疫吸附测定法(ELISA)检测各组大鼠血清中的前炎症细胞因子和抗炎症细胞因子,前炎症细胞因子包括白介素-1β(IL-1β)、白介素-8(IL-8)、肿瘤坏死因子-α(TNF-α),抗炎症细胞因子包括白介素-4(IL-4)、白介素-10(IL-10);大鼠子宫形态学观察后,免疫组织化学法检测各组大鼠子宫组织肿瘤坏死因子-α(TNF-α)、转化生长因子-β1(TGF-β1)、血管内皮生长因子(VEGF)、细胞间粘附分子-1(ICAM-1)、基质金属蛋白酶-2(MMP-2)的表达。
结果:慢性盆腔炎模型大鼠血清前炎症细胞因子IL-1β、IL-8、TNF-α水平较正常组升高,抗炎症细胞因子IL-4、IL-10水平较正常组降低,子宫组织中的粘连相关免疫分子TNF-α、TGF-β1、VEGF、ICAM-1、MMP-2表达均较正常组升高,差异有极显著性(P<0.01)。药物干预后,各用药组大鼠血清前炎症细胞因子水平均下降,抗炎症细胞因子水平均升高,子宫组织中的MMP-2表达较模型组升高,TNF-α、TGF-β1、VEGF、ICAM-1的表达均较模型组降低(P<0.05)。桂枝茯苓中剂量组对血清和组织中的TNF-α、TGF-β1及组织中ICAM-1、MMP-2的调节作用优于少腹逐瘀组和金刚藤组(P<0.05)。桂枝茯苓胶囊干预的盆腔炎模型大鼠的炎症细胞因子和粘连相关免疫分子之间存在着显著正负相关性。
结论:活血化瘀法、清热解毒法可通过对大鼠血清、子宫组织的炎症细胞因子和粘连相关免疫分子的调节,有效消除盆腔炎模型大鼠盆腔炎症,缓解盆腔粘连,改善机体免疫功能。活血化瘀法对粘连相关免疫分子表达的调节作用明显优于清热解毒法,其在改善慢性盆腔炎症粘连的免疫调节方面表现出优势。
Objective: To explore the immunoregulation mechanism of the treatment of activating blood to remove blood stasis on chronic pelvic inflammatory disease(CPID) by observing the level of inflammatory cytokines and adhesion related molecules in serum and uterine tissue of CPID model rats.
Method:Female Wistar rats were used and the CPID model was established with mixed bacteria injection combined with mechanic injury. Ninty-six rats were randomly divided into normal group, sham operation group, model group, Guizhi Fuling high, medium and low dose group, Shaofuzhuyu group and Jingangteng group. The histomorphological changes of the rats' uteri were observed using light microscope.The enzyme-linked immunosorbent assay (ELISA) was applied to measure the concentration of pro-inflammatory cytokines (IL-1β, IL-8, TNF-α) and anti-inflammatory cytokines(IL-4, IL-10)in serum of rats in each group; the immunohistochemistry method was used to detect the expression of the following indices in rats’uterine tissue: tumor necrosis factor-α(TNF-α), transforming growth factor-β1(TGF-β1), vascular endothelial growth factor(VEGF), intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase protein -2(MMP-2).
Results:The serous levels of pro-inflammatory cytokines (IL-1β, IL-8, TNF-α)in model group were significantly higher than those in normal group; the serous levels of anti-inflammatory cytokines (IL-4, IL-10) in model group were significantly lower than those in normal group. The expression of adhesion related immune molecules(TNF-α, TGF-β1, VEGF, ICAM-1, MMP-2) in model group was higher than normal group significantly(P<0.01).After treatment, the pro-inflammatory cytokine levels were decreased while the anti-inflammatory cytokine levels were elevated,the expression of TNF-α、TGF-β1、VEGF and ICAM-1 decreased, and MMP-2 elevated(P<0.05). Guizhi Fuling medium dose group showed abetter therapeutic effects on regulating TNF-α、TGF-β1、ICAM-1、MMP-2 than Shaofuzhuyu group and Jingangteng group(P<0.05). There were positive or negative correlation between Inflammatory cytokines and adhesion related immune molecules in Guizhi Fuling medium dose group.
Conclusions: The method of activating blood to remove blood stasis and clearing away heat and toxin could adjust the level of inflammatory cytokines and various immune-related adhesion molecule levels, thus adjust the immune balance effectively so as to inhibit inflammation, prevent and alleviate the pelvic adhesions. The method of activating blood to remove blood stasis is superior to the method of clearing away heat and toxin in the regulation of adhesion related immune molecules so it has an advantage of alleviating the adhesion in CPID through certain immune mechanisms.
引文
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