川芎嗪治疗膝骨性关节炎的临床观察及对兔关节软骨细胞增殖与凋亡的影响
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摘要
目的
1、观察川芎嗪治疗瘀血阻络型膝骨性关节炎的临床疗效,为川芎嗪应用于骨性关节炎的防治提供临床资料。
2、探讨川芎嗪对兔软骨细胞增殖与凋亡影响及其作用机制,从分子生物学水平阐明川芎嗪保护关节软骨的部分机理。方法
1、选择临床瘀血阻络型膝骨性关节炎患者76例(102个膝),用抽签法随机分为治疗组38例(52个膝)与对照组38例(50个膝)。治疗组于患侧膝关节腔内注射川芎嗪注射液,每次2m1,每周1次,连续5周为1个疗程。对照组于患侧膝关节腔内注射玻璃酸钠注射液,每次2m1,每周1次,连续5周为1个疗程。观察治疗前后患者的临床疗效、关节症状总积分、关节液中IL-1、IL-6、TNF-α、MMP-3含量变化。
2、4周龄雄性新西兰大白兔16只,取膝关节软骨建立体外培养的软骨细胞体系,甲苯胺蓝染色鉴定软骨细胞,荧光倒置相差显微镜观察软骨细胞的形态结构；MTT检测第2至5代软骨细胞的增长曲线。
3、第3代软骨细胞同步化,抽签法随机分为对照组(川芎嗪终浓度0ug/L的10%FBS DMEM)、中剂量组(川芎嗪终浓度50ug/L的10%FBS DMEM),分别培养24h、48h、72h、96h, MTT检测软骨细胞的活性,确定川芎嗪的有效干预时间。第3代软骨细胞同步化,抽签法随机分为对照组(川芎嗪终浓度0ug/L的10%FBS DMEM)、低剂量组(川芎嗪终浓度25ug/L的10%FBS DMEM),中剂量组(川芎嗪终浓度50ug/L的10%FBS DMEM)、高剂量组(川芎嗪终浓度100ug/L的10%FBS DMEM),各组干预72h后,透射电子显微镜观察软骨细胞的形态结构,流式细胞仪检测软骨细胞周期分布,RT-PCR检测软骨细胞CyclinD1、CDK4、p21mRNA表达,Western-Blot检测软骨细胞CyclinD1、CDK4、p21蛋白表达。
4、第3代软骨细胞培养72h,加入含SNP终浓度为lmmol/L的10%FBS DMEM干预24h复制软骨细胞凋亡模型,细胞分为模型组、低剂量组、中剂量组、高剂量组；并设正常组,加入10%FBS DMEM,分组进行干预12h、24h、36h, MTT检测软骨细胞的活性,确定川芎嗪的有效干预时间。第3代软骨细胞培养72h,正常组加入10%FBS DMEM,模型组、低剂量组、中剂量组、高剂量组加入含SNP终浓度为1mmol/L的10%FBS DMEM干预24h后,各组再加相应药物干预24h,MTT法检测软骨细胞活性,透射电子显微镜观察软骨细胞的形态结构,流式细胞仪检测软骨细胞的凋亡,RT-PCR检测软骨细胞Bcl-2、Bax mRNA表达,Western-Blot检测软骨细胞Bcl-2、Bax蛋白表达,比色法检测软骨细胞CasDase-3、CasDase-9蛋白的活性。
结果
1、川芎嗪治疗瘀血阻络型膝骨性关节炎的临床研究：76例(102个膝)患者,完成治疗73例(99个膝),其中,治疗组剔除1例(1个膝),对照组剔除2例(2个膝)。两组治疗后临床优良率、有效率比较无显著性差异(P>0.05)。治疗后两组症状积分前后分值差异比较,治疗组虽低于对照组,但无显著性差异(P>0.05)。与治疗前比较,两组IL-1、IL-6、 TNF-α、MMP-3含量均明显降低(P0.05)
2、原代、第2代、第3代软骨细胞生长状况良好,甲苯胺蓝染色后,胞浆内见紫红色异染颗粒。增殖曲线上,第2、3代软骨细胞生长旺盛,活性比第4、5代软骨细胞高,尤其是第3代软骨细胞；第3代软骨细胞,第3天增殖开始加速,第4天进入指数生长期,第5、6天增殖速度逐渐减慢。
3、川芎嗪促进软骨细胞增殖的研究：
(1)川芎嗪促进软骨细胞增殖的有效作用时间为72h。中剂量组干预72h细胞OD值明显高于干预24h、48h、96h(P<0.05)。
(2)透射电子显微镜观察软骨细胞的形态结构：干预72h后各组细胞呈圆形、椭圆形、多角形,核呈圆形为主,可见分叶状、不规则,染色质分布均匀,核仁清晰,中剂量组可见胞质中粗面内质网丰富扩张,高剂量组可见细胞核的分裂。
(3)软骨细胞的周期分布：干预72h,中剂量组、高剂量组G0/G1期显著低于对照组同期(P (4)软骨细胞CyclinD1、CDK4mRNA表达,干预72h,中剂量组、高剂量组明显高于对照组、低剂量组(P<0.05)；软骨细胞p21mRNA表达,干预72h,中剂量组、高剂量组明显低于对照组(P<0.05)。
(5)软骨细胞CyclinD1、CDK4蛋白表达,干预72h,中剂量组、高剂量组明显高于对照组、低剂量组(P<0.05)；软骨细胞p21蛋白表达,中剂量组、高剂量组明显低于对照组、低剂量组(P<0.05)。
4、川芎嗪抑制软骨细胞凋亡的研究：
(1)川芎嗪抑制软骨细胞凋亡的有效作用时间为24h；干预24h后,软骨细胞0D值,中剂量组、高剂量组明显高于模型组(P<0.05)。
(2)透射电子显微镜观察软骨细胞的形态结构：干预24h,模型组可见大量碎裂的坏死、凋亡的软骨细胞；低剂量组见较多碎裂、凋亡的软骨细胞；中剂量组、高剂量组见少量坏死、凋亡的软骨细胞。
(3)软骨细胞的总凋亡率：模型组(54.30±1.58)%,低剂量组(27.00±.3.24)%,中剂量组(19.09±.3.33)%,高剂量组(16.22±2.59)%,中剂量组、高剂量组明显低于模型组、低剂量组(P<0.05)。
(4)软骨细胞Bax mRNA表达,中剂量组、高剂量组明显低于模型组(P<0.05)；软骨细胞Bcl-2mRNA表达,中剂量组、高剂量组明显高于模型组(P<0.05)。
(5)软骨细胞Bax蛋白的表达,中剂量组、高剂量组明显低于模型组(P<0.05);软骨细胞Bcl-2蛋白的表达,中剂量组、高剂量组明显高于模型组(P<0.05)。
(6)软骨细胞Caspase-3、Caspase-9蛋白活性,中剂量组、高剂量组显著低于模型组(P<0.01),中剂量组、高剂量明显低于低剂量组(P<0.05)。
结论
1、川芎嗪关节腔注射治疗能有效缓解瘀血阻络型膝骨性关节炎患者的临床症状,降低关节液中IL-1、IL-6、TNF-α、MMP-3的含量,是治疗膝骨性关节炎的有效方法之一。
2、川芎嗪能促进软骨细胞增殖。其作用机制可能通过上调软骨细胞周期正向调节因子CyclinD1、CDK4表达,下调负向调节因子p21表达,加速软骨细胞周期关键限制点G1/S期的切换,促进软骨细胞周期的进程。
3、川芎嗪能抑制软骨细胞凋亡。其作用机制可能通过下调软骨细胞促凋亡因子Bax、 Caspase-3、Caspase-9的表达,上调抗凋亡因子Bcl-2表达,抑制软骨细胞线粒体凋亡通路的信号转导。
Objective
1、To observe the clinical effect of Ligustrazine on knee osteoarthritis (KOA) with syndrome of static blood blocking collaterals, and provide clinical data for the treatment of KOA.
2、To explore the effects of Ligustrazine on proliferation and apoptosis of rabbits chondrocyte, and illuminate some mechanism of Ligustrazine protecting articular cartilage on the molecular biology level.
Methods
1、76KOA patients with syndrome of static blood blocking collaterals were randomly assigned to treatment group (n=38,52knees), control group (n=38,50knees). The treatment group received intraarticular injection of2mL ligustrazine hydrochloride injection on the affected knee joint,1time a week,5weeks as a course of treatment, and the control group received sodium hyaluronate injection. The curative effect, total integral of joint symptoms, and the contents of IL-1, IL-6, TNF-α and MMP-3were observed before and after the treatment.
2、Cartilage was isolated from the knee joint of16male New Zealand white rabbits of4-week old and used to establish cultured primary chondrocytes. Chondrocytes were identified with toluidine blue stain, morphosis was observed by fluorescent inverted phase contrast microscope, and growth curves of the2nd to5th generation chondrocytes were detected with MTT method.
3、The3rd generation chondrocytes were synchronized, then randomly divided inio control group and middle dose group, which were added10%FBS DMEM containing Ligustrazine final concentration of0ug/L and50ug/L respectively, and cultured for24h,48h,72h and96h, the activity of chondrocytes through MTT to determine effective intervention time of Ligustrazine. The3rd generation chondrocytes were synchronized, then randomly divided inio control group, low dose group, middle dose group and high dose group, which were added10%FBS DMEM containing Ligustrazine final concentration of0ug/L,25ug/L,50ug/L and100ug/L respectively, and cultured for72h, chondrocytes morphology were observed under transmission electron microscopy, cycle distribution of chondrocytes were detected with flow cytometry, the mRNA expression of CyclinDl, CDK4and p21by RT-PCR, and the protein expression by western-blot.
4、The3rd generation chondrocytes were cultured for72h, then treated with10%FBS DMEM containing SNP final concentration of1mmol/L, and cultured for24h to duplicate models of chondrocytes apoptosis. Then chondrocytes were randomly divided into model group, low dose group, middle dose group and high dose group, and the normal group as control. Each group was added10%FBS DMEM, and cultured for12h,24h,36h, the activity of chondrocytes through MTT to determine effective intervention time of Ligustrazine. The3rd generation chondrocytes were cultured for72h, and the normal group was added10%FBS DMEM. The model group, low dose group, middle dose group and high dose group were added10%FBS DMEM containing SNP final concentration of1mmol/L for24h, then each group was added corresponding drugs for24h. The activity of chondrocytes through MTT, chondrocytes morphology were observed under transmission electron microscopy, chondrocytes apoptosis was detected with flow cytometry, the mRNA expression of Bcl-2and Bax by RT-PCR, and the protein expression of Bcl-2and Bax by western-blot, and the activity of Caspase-3and Caspase-9were measured using colorimeters.
Results
1、The clinical research results showed that76KOA patients (102knees) with syndrome of static blood blocking collaterals were collected, and73patients (99knees) completed the treatment. Among them,1patients (1knees) fell off in treatment group, and2patients (2knees) fell off in control group. After the treatment, there was no significant difference in the excellent and good rate and effective rate between two groups (P>0.05); the decreasing amplitude of integral of joint symptoms in treatment group was lower than that in control group, and there was no significant difference between them (P>0.05); the contents of IL-1, IL-6, TNF-α and MMP-3were significantly decreased in both groups (P<0.05); There was a difference in the decreasing amplitude of each index between the two groups after the treatment, but the difference was not significant.
2、The growing status of chondrocytes was good, the identification with toluidine blue staining, the cytoplasm was purple metachromatic granules in the original generation,2nd generation,3rd generation. In growth curve, the activity of2nd generation,3rd generation was better than that of4th generation,5th generation, especially for the3rd generation. The proliferation of the3rd generation was accelerated on the3rd day, then the chondrocytes enter exponential growth phase on the4th day, and the proliferation was slowed down on the5th and6th day gradually.
3、The research of Ligustrazine promoting the proliferation of chondrocytes:
(1) Ligustrazine promote chondrocytes proliferation in effective time of72h. The chondrocytes OD values of middle dose group and high dose group were significantly higher than control group (P<0.05).
(2) The chondrocytes morphology were observed under transmission electron microscopy: After72h intervention, round, ellipse or polygon cells were found in each group, the nucleus appeared round, lobulated and irregular shape, the chromatin was well-distributed, nucleolus was clear, dilated rough endoplasmic reticulum was found in middle dose group, and the nuclear division appeared in high dose group.
(3) The cycle distribution of chondrocyte:After72h intervention, the G0/G1phase of middle and high dose groups was respectively lower than control group (P<0.01), the G2/M phase was respectively higher than control group (P<0.05), and PI was respectively higher than control group (P<0.05).
(4) The mRNA expression of CyclinDl, CDK4in chondrocytes of middle dose group and high dose group were significantly higher than control group (P<0.05); the expression of p21mRNA of middle dose group and high dose group were significantly lower than control group (P<0.05).
(5) The protein expression of CyclinD1, CDK4in chondrocytes of middle dose group and high dose group were significantly higher than control group (P<0.05); the expression of p21protein of middle dose group and high dose group were significantly lower than control group (P<0.05).4、The research of Ligustrazine inhibiting the apoptosis of chondrocytes:
(1) Effective intervention time of Ligustrazine inhibiting chondrocyte apoptosis was24h. After24h intervention, the chondrocytes OD values of middle dose group and high dose group were significantly higher than model group (P<0.05).
(2)The chondrocytes morphology were observed under transmission electron microscopy:After72h intervention, lots of chondrocytes apoptosis or fragmentation were observed in model group, more chondrocytes apoptosis or fragmentation were found in low dose group, and the less chondrocytes apoptosis or fragmentation appeared in high dose group.
(3) The chondrocytes apoptosis rate of model group, low dose group, middle dose group and high dose group were (54.30±1.58)%,(27.00±3.24)%,(19.09±3.33)%and (16.22±2.59)%respectively. The chondrocytes apoptosis rate of middle dose group, high dose group was respectively lower than model group, low dose group (P<0.05).
(4) The expression of Bax mRNA of middle dose group, high dose group was significantly lower than model group (P<0.05). The expression of Bcl-2mRNA of middle dose group, high dose group was respectively higher than model group (P<0.05).
(5) The expression of Bax protein of middle dose group, high dose group was respectively lower than model group (P<0.05). The expression of Bcl-2protein of middle dose group, high dose group was respectively higher than model group (P<0.05).
(6) The protein activity of Caspase-3, Caspase-9in chondrocytes of middle dose group, high dose group was respectively lower than model group (P<0.05), and also significantly lower than low dose group (P<0.05).
Conclusions
1. Intraarticular injection of ligustrazine can relieve the clinical symptoms of KOA patients with syndrome of static blood blocking collaterals effectively, decrease the contents of IL-1, IL-6, TNF-α and MMP-3in synovia, and the method has good effect on the treatment of KOA.
2. Ligustrazine can promote the proliferation of chondrocytes, and its mechanism is probably related with the effect of up-regulating expression of chondrocyte cycle positive regulators, including CyclinD1and CDK4, inhibiting negative regulator p21, activating the key point of switch from G1phase to S Phase during chondrocyte cycle, and promoting the process of chondrocytes cycle.
3. Ligustrazine can inhibit the apoptosis of chondrocytes, and its mechanism is probably related with the effect of down-regulating expression of chondrocyte pro-apoptosis gene, including Bax, caspase-3and caspase-9, up-regulating expression of anti-apoptosis gene Bcl-2, and inhibiting chondrocyte mitochondrial signal transduction pathway of apoptosis.
1、观察川芎嗪治疗瘀血阻络型膝骨性关节炎的临床疗效,为川芎嗪应用于骨性关节炎的防治提供临床资料。
2、探讨川芎嗪对兔软骨细胞增殖与凋亡影响及其作用机制,从分子生物学水平阐明川芎嗪保护关节软骨的部分机理。方法
1、选择临床瘀血阻络型膝骨性关节炎患者76例(102个膝),用抽签法随机分为治疗组38例(52个膝)与对照组38例(50个膝)。治疗组于患侧膝关节腔内注射川芎嗪注射液,每次2m1,每周1次,连续5周为1个疗程。对照组于患侧膝关节腔内注射玻璃酸钠注射液,每次2m1,每周1次,连续5周为1个疗程。观察治疗前后患者的临床疗效、关节症状总积分、关节液中IL-1、IL-6、TNF-α、MMP-3含量变化。
2、4周龄雄性新西兰大白兔16只,取膝关节软骨建立体外培养的软骨细胞体系,甲苯胺蓝染色鉴定软骨细胞,荧光倒置相差显微镜观察软骨细胞的形态结构；MTT检测第2至5代软骨细胞的增长曲线。
3、第3代软骨细胞同步化,抽签法随机分为对照组(川芎嗪终浓度0ug/L的10%FBS DMEM)、中剂量组(川芎嗪终浓度50ug/L的10%FBS DMEM),分别培养24h、48h、72h、96h, MTT检测软骨细胞的活性,确定川芎嗪的有效干预时间。第3代软骨细胞同步化,抽签法随机分为对照组(川芎嗪终浓度0ug/L的10%FBS DMEM)、低剂量组(川芎嗪终浓度25ug/L的10%FBS DMEM),中剂量组(川芎嗪终浓度50ug/L的10%FBS DMEM)、高剂量组(川芎嗪终浓度100ug/L的10%FBS DMEM),各组干预72h后,透射电子显微镜观察软骨细胞的形态结构,流式细胞仪检测软骨细胞周期分布,RT-PCR检测软骨细胞CyclinD1、CDK4、p21mRNA表达,Western-Blot检测软骨细胞CyclinD1、CDK4、p21蛋白表达。
4、第3代软骨细胞培养72h,加入含SNP终浓度为lmmol/L的10%FBS DMEM干预24h复制软骨细胞凋亡模型,细胞分为模型组、低剂量组、中剂量组、高剂量组；并设正常组,加入10%FBS DMEM,分组进行干预12h、24h、36h, MTT检测软骨细胞的活性,确定川芎嗪的有效干预时间。第3代软骨细胞培养72h,正常组加入10%FBS DMEM,模型组、低剂量组、中剂量组、高剂量组加入含SNP终浓度为1mmol/L的10%FBS DMEM干预24h后,各组再加相应药物干预24h,MTT法检测软骨细胞活性,透射电子显微镜观察软骨细胞的形态结构,流式细胞仪检测软骨细胞的凋亡,RT-PCR检测软骨细胞Bcl-2、Bax mRNA表达,Western-Blot检测软骨细胞Bcl-2、Bax蛋白表达,比色法检测软骨细胞CasDase-3、CasDase-9蛋白的活性。
结果
1、川芎嗪治疗瘀血阻络型膝骨性关节炎的临床研究：76例(102个膝)患者,完成治疗73例(99个膝),其中,治疗组剔除1例(1个膝),对照组剔除2例(2个膝)。两组治疗后临床优良率、有效率比较无显著性差异(P>0.05)。治疗后两组症状积分前后分值差异比较,治疗组虽低于对照组,但无显著性差异(P>0.05)。与治疗前比较,两组IL-1、IL-6、 TNF-α、MMP-3含量均明显降低(P
2、原代、第2代、第3代软骨细胞生长状况良好,甲苯胺蓝染色后,胞浆内见紫红色异染颗粒。增殖曲线上,第2、3代软骨细胞生长旺盛,活性比第4、5代软骨细胞高,尤其是第3代软骨细胞；第3代软骨细胞,第3天增殖开始加速,第4天进入指数生长期,第5、6天增殖速度逐渐减慢。
3、川芎嗪促进软骨细胞增殖的研究：
(1)川芎嗪促进软骨细胞增殖的有效作用时间为72h。中剂量组干预72h细胞OD值明显高于干预24h、48h、96h(P<0.05)。
(2)透射电子显微镜观察软骨细胞的形态结构：干预72h后各组细胞呈圆形、椭圆形、多角形,核呈圆形为主,可见分叶状、不规则,染色质分布均匀,核仁清晰,中剂量组可见胞质中粗面内质网丰富扩张,高剂量组可见细胞核的分裂。
(3)软骨细胞的周期分布：干预72h,中剂量组、高剂量组G0/G1期显著低于对照组同期(P
(5)软骨细胞CyclinD1、CDK4蛋白表达,干预72h,中剂量组、高剂量组明显高于对照组、低剂量组(P<0.05)；软骨细胞p21蛋白表达,中剂量组、高剂量组明显低于对照组、低剂量组(P<0.05)。
4、川芎嗪抑制软骨细胞凋亡的研究：
(1)川芎嗪抑制软骨细胞凋亡的有效作用时间为24h；干预24h后,软骨细胞0D值,中剂量组、高剂量组明显高于模型组(P<0.05)。
(2)透射电子显微镜观察软骨细胞的形态结构：干预24h,模型组可见大量碎裂的坏死、凋亡的软骨细胞；低剂量组见较多碎裂、凋亡的软骨细胞；中剂量组、高剂量组见少量坏死、凋亡的软骨细胞。
(3)软骨细胞的总凋亡率：模型组(54.30±1.58)%,低剂量组(27.00±.3.24)%,中剂量组(19.09±.3.33)%,高剂量组(16.22±2.59)%,中剂量组、高剂量组明显低于模型组、低剂量组(P<0.05)。
(4)软骨细胞Bax mRNA表达,中剂量组、高剂量组明显低于模型组(P<0.05)；软骨细胞Bcl-2mRNA表达,中剂量组、高剂量组明显高于模型组(P<0.05)。
(5)软骨细胞Bax蛋白的表达,中剂量组、高剂量组明显低于模型组(P<0.05);软骨细胞Bcl-2蛋白的表达,中剂量组、高剂量组明显高于模型组(P<0.05)。
(6)软骨细胞Caspase-3、Caspase-9蛋白活性,中剂量组、高剂量组显著低于模型组(P<0.01),中剂量组、高剂量明显低于低剂量组(P<0.05)。
结论
1、川芎嗪关节腔注射治疗能有效缓解瘀血阻络型膝骨性关节炎患者的临床症状,降低关节液中IL-1、IL-6、TNF-α、MMP-3的含量,是治疗膝骨性关节炎的有效方法之一。
2、川芎嗪能促进软骨细胞增殖。其作用机制可能通过上调软骨细胞周期正向调节因子CyclinD1、CDK4表达,下调负向调节因子p21表达,加速软骨细胞周期关键限制点G1/S期的切换,促进软骨细胞周期的进程。
3、川芎嗪能抑制软骨细胞凋亡。其作用机制可能通过下调软骨细胞促凋亡因子Bax、 Caspase-3、Caspase-9的表达,上调抗凋亡因子Bcl-2表达,抑制软骨细胞线粒体凋亡通路的信号转导。
Objective
1、To observe the clinical effect of Ligustrazine on knee osteoarthritis (KOA) with syndrome of static blood blocking collaterals, and provide clinical data for the treatment of KOA.
2、To explore the effects of Ligustrazine on proliferation and apoptosis of rabbits chondrocyte, and illuminate some mechanism of Ligustrazine protecting articular cartilage on the molecular biology level.
Methods
1、76KOA patients with syndrome of static blood blocking collaterals were randomly assigned to treatment group (n=38,52knees), control group (n=38,50knees). The treatment group received intraarticular injection of2mL ligustrazine hydrochloride injection on the affected knee joint,1time a week,5weeks as a course of treatment, and the control group received sodium hyaluronate injection. The curative effect, total integral of joint symptoms, and the contents of IL-1, IL-6, TNF-α and MMP-3were observed before and after the treatment.
2、Cartilage was isolated from the knee joint of16male New Zealand white rabbits of4-week old and used to establish cultured primary chondrocytes. Chondrocytes were identified with toluidine blue stain, morphosis was observed by fluorescent inverted phase contrast microscope, and growth curves of the2nd to5th generation chondrocytes were detected with MTT method.
3、The3rd generation chondrocytes were synchronized, then randomly divided inio control group and middle dose group, which were added10%FBS DMEM containing Ligustrazine final concentration of0ug/L and50ug/L respectively, and cultured for24h,48h,72h and96h, the activity of chondrocytes through MTT to determine effective intervention time of Ligustrazine. The3rd generation chondrocytes were synchronized, then randomly divided inio control group, low dose group, middle dose group and high dose group, which were added10%FBS DMEM containing Ligustrazine final concentration of0ug/L,25ug/L,50ug/L and100ug/L respectively, and cultured for72h, chondrocytes morphology were observed under transmission electron microscopy, cycle distribution of chondrocytes were detected with flow cytometry, the mRNA expression of CyclinDl, CDK4and p21by RT-PCR, and the protein expression by western-blot.
4、The3rd generation chondrocytes were cultured for72h, then treated with10%FBS DMEM containing SNP final concentration of1mmol/L, and cultured for24h to duplicate models of chondrocytes apoptosis. Then chondrocytes were randomly divided into model group, low dose group, middle dose group and high dose group, and the normal group as control. Each group was added10%FBS DMEM, and cultured for12h,24h,36h, the activity of chondrocytes through MTT to determine effective intervention time of Ligustrazine. The3rd generation chondrocytes were cultured for72h, and the normal group was added10%FBS DMEM. The model group, low dose group, middle dose group and high dose group were added10%FBS DMEM containing SNP final concentration of1mmol/L for24h, then each group was added corresponding drugs for24h. The activity of chondrocytes through MTT, chondrocytes morphology were observed under transmission electron microscopy, chondrocytes apoptosis was detected with flow cytometry, the mRNA expression of Bcl-2and Bax by RT-PCR, and the protein expression of Bcl-2and Bax by western-blot, and the activity of Caspase-3and Caspase-9were measured using colorimeters.
Results
1、The clinical research results showed that76KOA patients (102knees) with syndrome of static blood blocking collaterals were collected, and73patients (99knees) completed the treatment. Among them,1patients (1knees) fell off in treatment group, and2patients (2knees) fell off in control group. After the treatment, there was no significant difference in the excellent and good rate and effective rate between two groups (P>0.05); the decreasing amplitude of integral of joint symptoms in treatment group was lower than that in control group, and there was no significant difference between them (P>0.05); the contents of IL-1, IL-6, TNF-α and MMP-3were significantly decreased in both groups (P<0.05); There was a difference in the decreasing amplitude of each index between the two groups after the treatment, but the difference was not significant.
2、The growing status of chondrocytes was good, the identification with toluidine blue staining, the cytoplasm was purple metachromatic granules in the original generation,2nd generation,3rd generation. In growth curve, the activity of2nd generation,3rd generation was better than that of4th generation,5th generation, especially for the3rd generation. The proliferation of the3rd generation was accelerated on the3rd day, then the chondrocytes enter exponential growth phase on the4th day, and the proliferation was slowed down on the5th and6th day gradually.
3、The research of Ligustrazine promoting the proliferation of chondrocytes:
(1) Ligustrazine promote chondrocytes proliferation in effective time of72h. The chondrocytes OD values of middle dose group and high dose group were significantly higher than control group (P<0.05).
(2) The chondrocytes morphology were observed under transmission electron microscopy: After72h intervention, round, ellipse or polygon cells were found in each group, the nucleus appeared round, lobulated and irregular shape, the chromatin was well-distributed, nucleolus was clear, dilated rough endoplasmic reticulum was found in middle dose group, and the nuclear division appeared in high dose group.
(3) The cycle distribution of chondrocyte:After72h intervention, the G0/G1phase of middle and high dose groups was respectively lower than control group (P<0.01), the G2/M phase was respectively higher than control group (P<0.05), and PI was respectively higher than control group (P<0.05).
(4) The mRNA expression of CyclinDl, CDK4in chondrocytes of middle dose group and high dose group were significantly higher than control group (P<0.05); the expression of p21mRNA of middle dose group and high dose group were significantly lower than control group (P<0.05).
(5) The protein expression of CyclinD1, CDK4in chondrocytes of middle dose group and high dose group were significantly higher than control group (P<0.05); the expression of p21protein of middle dose group and high dose group were significantly lower than control group (P<0.05).4、The research of Ligustrazine inhibiting the apoptosis of chondrocytes:
(1) Effective intervention time of Ligustrazine inhibiting chondrocyte apoptosis was24h. After24h intervention, the chondrocytes OD values of middle dose group and high dose group were significantly higher than model group (P<0.05).
(2)The chondrocytes morphology were observed under transmission electron microscopy:After72h intervention, lots of chondrocytes apoptosis or fragmentation were observed in model group, more chondrocytes apoptosis or fragmentation were found in low dose group, and the less chondrocytes apoptosis or fragmentation appeared in high dose group.
(3) The chondrocytes apoptosis rate of model group, low dose group, middle dose group and high dose group were (54.30±1.58)%,(27.00±3.24)%,(19.09±3.33)%and (16.22±2.59)%respectively. The chondrocytes apoptosis rate of middle dose group, high dose group was respectively lower than model group, low dose group (P<0.05).
(4) The expression of Bax mRNA of middle dose group, high dose group was significantly lower than model group (P<0.05). The expression of Bcl-2mRNA of middle dose group, high dose group was respectively higher than model group (P<0.05).
(5) The expression of Bax protein of middle dose group, high dose group was respectively lower than model group (P<0.05). The expression of Bcl-2protein of middle dose group, high dose group was respectively higher than model group (P<0.05).
(6) The protein activity of Caspase-3, Caspase-9in chondrocytes of middle dose group, high dose group was respectively lower than model group (P<0.05), and also significantly lower than low dose group (P<0.05).
Conclusions
1. Intraarticular injection of ligustrazine can relieve the clinical symptoms of KOA patients with syndrome of static blood blocking collaterals effectively, decrease the contents of IL-1, IL-6, TNF-α and MMP-3in synovia, and the method has good effect on the treatment of KOA.
2. Ligustrazine can promote the proliferation of chondrocytes, and its mechanism is probably related with the effect of up-regulating expression of chondrocyte cycle positive regulators, including CyclinD1and CDK4, inhibiting negative regulator p21, activating the key point of switch from G1phase to S Phase during chondrocyte cycle, and promoting the process of chondrocytes cycle.
3. Ligustrazine can inhibit the apoptosis of chondrocytes, and its mechanism is probably related with the effect of down-regulating expression of chondrocyte pro-apoptosis gene, including Bax, caspase-3and caspase-9, up-regulating expression of anti-apoptosis gene Bcl-2, and inhibiting chondrocyte mitochondrial signal transduction pathway of apoptosis.
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