前列宁通过调控前列腺MMP-2、TIMP-2降解层粘连蛋白治疗良性前列腺增生的机制研究

Mechanism of Qianliening in Treatment of Benign Prostatic Hyperplasia by Regulating MMP-2 and TIMP-2 Degraded Laminin in Prostate histiocyte

目的探讨前列宁(QLN)通过调控前列腺MMP-2、TIMP-2降解层粘连蛋白(LN)治疗良性前列腺增生的机制。方法将50只SPF级6~7周龄雄性SD大鼠采用随机数字表法分为空白组,模型组和低、中、高剂量组,每组10只,除空白组外,其余组采用氯胺酮腹腔注射麻醉去势摘除双侧睾丸、1周后按5 mg/kg皮下注射丙酸睾酮造模,共28 d,造模期间,空白组、模型组按10 ml/(kg·d)灌胃生理盐水,低、中、高剂量组分别按3 g、6 g、12 g/(kg·d)灌胃QLN,1次/d,连续28 d。末次灌胃后禁食12 h,分离各组大鼠完整的前列腺组织并计算前列腺体积、前列腺湿重及前列腺指数(PI),免疫组化检测各组大鼠前列腺组织LN表达水平;取对数生长期的人前列腺增生细胞-1(BPH-1)分为空白组和低、中、高剂量组,分别按浓度为0、0.25、0.5、1.0 mg/mL QLN干预48 h,倒置显微镜观察各组细胞形态,CCK-8法检测各组细胞活性,Western blot分别检测各组细胞MMP-2、TIMP-2、LN蛋白表达量。结果与空白组比较,模型组大鼠前列腺体积、前列腺湿重及PI均明显增大(P<0.01),前列腺组织LN表达水平均明显增强(P<0.05);与模型组比较,低、中、高剂量组大鼠前列腺体积、前列腺湿重及PI均明显减小(P<0.05),低、中、高剂量组大鼠前列腺组织LN表达水平均不同程度降低(P<0.05)。与空白组比较,中、高剂量组BPH-1细胞随着浓度的增高,由菱形变成不规则,密度明显减少,生长速度缓慢,24、48 h BPH-1细胞活力均明显下降(P<0.05),MMP-2、LN蛋白表达量均明显降低,TIMP-2蛋白表达量明显增高(P均<0.05)。结论QLN能有效减小前列腺体积、前列腺湿重及PI,降低前列腺组织LN表达水平,其机制之一可能是通过调控前列腺组织及BPH-1中MMP-2、TIMP-2降解LN而得以实现。

Objective:To observe the regulatory effect of QLN on matrix metalloproteinase-2(MMP-2)and tissue inhibitors metalloproteinase-2(TIMP-2)mediated laminin(LN),and explore the therapeutic mechanism of QLN on benign prostatic hyperplasia(BPH).Methods:Fifty adult male SD rats were randomly divided into blank group,model group,low-does,medium-does and highdoes treatment group,with 10 rats in each group.Except for the blank group,the model group and the low,medium,and high treatment groups were anesthetized with ketamine intraperitoneal injection to remove both testicles.After one week,each rat was subcutaneously injected with testosterone propionate(5 mg/kg)for a total of 28 days.During the modeling period,the blank and model group were fed with regular feed and gavage of physiological saline(10 mL/kg·d-1),QLN low(3 g/kg·d-1),medium(6 g/kg·d-1),and high(12 g/kg·d-1)were administered by gavage once a day for 28 consecutive days.After the last administration,rats in each group fasted for 12 hours to separate intact prostate tissue.The wet weight and volume of the prostate in each group were measured,and the prostate index(PI:wet weight of the prostate/rat body weight)was calculated.The expression of LN in prostate tissue was observed by immunohistochemistry;Human prostate hyperplasia cells(BPH-1)were cultured in vitro and divided into blank group,low,medium,and high-dose groups,which were administered QLN 0 mg/mL,0.25 mg/mL,0.5 mg/mL,1.0 mg/mL respectively for 48 hours.The cell morphology of each group was observed under inverted microscope,and the cell viability of each group was detected using CCK-8 method.The expression of MMP2,TIMP-2,and LN proteins was detected using Western blot.Results:Animal experiments showed that the prostate volume,prostate wet weight,and prostate index of the model group significantly increased(P<0.05)compared to the blank group,indicating successful modeling.Compared with the model group,the prostate volume,wet weight,and prostate index of rats in the low-does,medium-does,and high-dose treatment groups significantly reduced(P<0.05).Immunohistochemistry showed that compared with the blank group,the expression of LN in the prostate of rats in the model group significantly increased(P<0.05).Compared with the model group,the expression of LN in the prostate of rats in the low-does,medium-does,and high-dose treatment groups reduced to varying degrees(P<0.05).Cell experiments showed that compared with the blank group(QLN 0 mg/mL),the medium(0.5 mg/mL)and high dose(1.0 mg/mL)groups showed significant decrease in BPH-1 cell density and slower cell growth rate under the microscope;significantly decreased BPH-1 cell viability at 24 and 48 hours with CCK-8 method(P<0.05);significantly decreased MMP-2 and LN protein expression,significantly increased TIMP-2 protein expression(P<0.05)with Western blot.Conclusion:QLN has a significant therapeutic effect on BPH,effectively reducing prostate tissue LN,prostate volume,prostate wet weight,and prostate index.One of the mechanisms may be achieved by regulating the degradation of LN by MMP-2 and TIMP-2.