| Objectives Reproductive toxicity is one of the clinical bottlenecks in the treatment of rheumatoid arthritis(RA)with tripterygium glycosides(TG).This study was to explore the effect of TG on the ovarian function of collagen-induced arthritis(CIA)in different time and time,and to observe the protective effect of Cuscuta chinensis extract on ovarian function of CIA rats,Which provides the experimental basis for TG for time treatment of RA.Methods 1.Rat feeding and selection 12 weeks old healthy female SD rats were paced in time biology laboratory,to adapt to a week after the experiment.After 5 days to do vaginal smear,select the emotional cycle of the rats into the experimental group,a total of 96.80 rats were used to establish CIA model,the same time,set the normal control rats 16.The model was successfully divided into CIA model group,TG day administration group,TG night administration group TG day administration and dodder extract,and the rats were randomly divided into CIA model group,TG day administration group,TG night administration group,Group,TG night administration + dodder extract group.Blank group and CIA model group were given normal saline,TG daily administration groupdaily 9am to TG 50 mg / kg.d,TG night administration group 9pm daily TG 50 mg / kg.d,continuous administration of 14 d.After the withdrawal of TG + dodder administration group daily administration of dodder extract 0.9g / kg.Bid,for 3 days.2.The rat estrus cycle was observed by rat vaginal exfoliative cell smear examination.3.The change of swelling degree of feet was observed by volumetric method(drainage method).4.15 days after the intervention,the use of MINRAY type X-ray machine on the rat ankle,foot beat X-ray film.5.On the 15 th day after the intervention,ZT2,ZT6,ZT10,ZT14,ZT18 and ZT22 were collected at 6 different time points.6.The levels of serum estradiol(E2),follicle stimulating hormone(FSH)and luteinizing hormone(LH)were measured by radioimmunoassay.7.Calculate ovary index.8.The levels of interleukin-6(IL-6),interleukin-1β(interleukin-1β),CLOCK and BMAL1 were detected by immunohis to chemistry.9.Statistical Methods All normal distribution measures were expressed as mean± standard(`x ± s).The difference between the two groups was judged by the variance analysis of multiple samples.The difference between the two groups was judged by t test.Using Spss17.0 software statistical analysis,test the level of α = 0.05.Results 1.Changes of estrous cycle in rats.The mean estrous cycle of the blank group and CIA model group was 4~5 d / time.TG was prolonged from 6~7 d / day in the daytime administration group and the night administration group Dodder extract group,the estrous cycle is 5~6d / times.2.Changes of serum E2,LH and FSH levels in rats.Compared with the blank group.the levels of serum E2,LH and FSH in the model group were not significantly different(P> 0.05),the level of serum E2 in TG group and TG + dodder extract group was significantly lower than that in blank group and model group(P <0.05).The level of LH secretion was significantly higher than that of blank group and model group(P <0.05),the level of serum FSH in TG group was significantly higher than that in blank group and model group(P <0.05),there was no significant difference in serum FSH level between blank group and model group(P> 0.05),Compared with TG group the levels of serum E2 in TG + dodder extract were significantly increased(P <0.05),serum FSH secretion decreased(P <0.05),LH was not significantly different(P> 0.05);Compared with the TG group the level of serum E2 in TG rats was higher(P <0.05),FSH secretion level is low but the difference is not obvious(P> 0.05),Serum LH secretion was lower(P <0.05);Compared with the control group,the level of E2 secretion was higher(P <0.05),and the levels of serum secreted by TG and dodder extract were significantly higher than those of the control group(P <0.05),FSH secretion level is low but the difference is not obvious(P>0.05),,Serum LH secretion was lower(P <0.05).3.The changes of serum E2,LH and FSH levels were observed day and night The serum samples of each group showed a certain rhythm changes,E2 showed a trend of day and night high,LH,FSH showed a trend of day and night low.In the case,E2 reaches the peak at ZT22,reaches the valley at ZT10;FSH reaches the peak at ZT18,reaches the valley at ZT14;when the peak of ZH occurs at ZT10,the valley occurs at ZT22.TG after the administration,they are lost the original rhythm.4.Ovarian index in each group Compared with the blank group there was no significant difference in ovarian index between the model group(P> 0.05).After administration,the ovary index of TG group and TG + dodder extract group was significantly lower than that of blank group(P <0.05);compared with TG group,there was no significant difference in ovarian index between TG + dodder extract(P> 0.05);compared with TG group,the ovarian index was higher in TG rats(P <0.05);compared with the control group the ovariectomized group was significantly higher than that of the control group(P <0.05).5.Changes of joint swelling in rats In addition to blank group,with the extension of modeling time,joint swelling gradually increased,double hind foot swelling gradually increased,then slowly reduced.Compared with the blank group,the difference was significant(P<0.05);compared with the model group,the swelling degree of TG group and TG + dodder extract group was significantly decreased(P <0.05);compared with TG day group,the swelling of TG group was significantly decreased(P <0.05);compared with the control group,the swelling degree of TG group and dodder extract group was significantly lower than that of TG group(P <0.05).6.Evaluation of joint imaging The ankle joint of the blank group was observed in the ankle joint,and the articular surface of the metacarpop halangeal joint was clear and smooth.There was no abnormality in the joint space.In the model group,the ankle joint was blurred and had obvious bone destruction.The joint space became narrowed or disappeared,and the swelling of the surrounding soft tissue was thickened.In the daytime group,the ankle joint was destroyed and blurred.Some of the joints were found to be bone defect.TG rats in the evening group also showed different degree of destruction,which was slightly lighter than that of the day group and the swelling degree of the soft tissue around the soft tissue was obviously reduced.7.HE staining of ovarian tissue Blank group and model group ovary tissue structure is normal,bulky,follicular growth is active,visible follicles at all levels,mature follicles and corpus luteum.Mature follicles in the granular cell level,follicular fluid-rich,good development of corpus luteum.The ovariectomized group was divided into two groups: fibrous connective tissue and adipose tissue.Most of the follicular follicles were reduced in the tissues,and the follicles were significantly reduced in the tissues.Reduced number of corpus luteum.TG group was lower than the blank group,the follicles were reduced at all levels,and lymphocyte infiltration was found in some follicles.The ovariectomized group of dodder extract was slightly smaller than the blank group,the lesion was mild and the lymphocyte infiltration was small.8.Immunohistochemical test results The HE staining of the ankle joint in the blank group showed smooth synovial membrane,1-2 layer synovial cells,no papillary processes or hyperplasia,no multinucleated cell infiltration.In the model group,the anastomosis of the CIA rats showed that the cartilage was damaged,the synovial cell hyperplasia was serious,the papillae were raised,and a lot of inflammatory cells were infiltrated.In the daytime administration group,synovial cell hyperplasia,papillae bulge and extensive inflammatory cell infiltration were observed in the ankle joint of rats.TG rats were treated with nocturnal administration of synovial hyperplasia,mastoid bulge,a small amount of inflammatory cell infiltration.The expression of IL-1β,IL-6,CLOCK and BMAL1 in the synovial cells of the model group were significantly higher than those in the blank group.Conclusions 1 There were significant differences in serum E2,LH and FSH levels between normal and CIA models,and the original rhythm was lost after TG administration.2 The effect of TG on the ovarian function of female CIA rats was different,and the damage of ovarian function in female CIA rats was different at different time.3 The ovarian dysfunction of female CIA rats may be related to the expression of CLOCK and BMAL1.4 Cuscuta extract had a protective effect on ovarian function damage induced by TG in CIA rats. |
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