| Objective1、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.Methods1、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.Results1、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).Conclusions1. 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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