| Objectives On the basis of circadian rhythm of CIA rats of inflammatory cytokines, we further observe the effect of TG on CIA rats inflammatory factor of circadian rhythm. And we study the treatment duration of MTX?LEF and TG on CIA rats. Then explore its mechanism. Methods 1 Experimental classification and the treatment 1.1 Part one: 80 female Wistar rats were placed in time biology laboratory and, according to a fixed light(L): a dark(d) time(L: D = 12: 12, light period for 07: 00 to 19: 00 dark period for 19: 00 ~ 07: 00).Rats were randomly divided into blank group and model group. 30 rats were in blank group. And 50 rats were in model group. Raised 10 days later, the rats were established of collagen induced arthritis(CIA) model. Then we should observe model rats and mark the rat ankle and foot swelling degree. In 42 d, blood was taken at different time(ZT2,6,10,14,18,22 hour). 1.2 Part two: 100 female Wistar rats were placed in time biology laboratory and, according to a fixed light(L): a dark(d) time(L: D = 12: 12, light period for 07: 00 to 19: 00 dark period for 19: 00 ~ 07: 00). Rats were randomly divided into TG-ZT6 group and TG-ZT18 group. 50 rats were in TG-ZT6 group. And 50 rats were in TG-ZT18 group. Raised 10 days later, the rats were established of collagen induced arthritis(CIA) model. Then we should observe model rats and mark the rat ankle and foot swelling degree. TG(40mg/kg) was administered orally at ZT6 or ZT18 every day for 42 d. In 42 d, blood was taken at different time(ZT2,6,10,14,18,22 hour). 1.3 Part three: 120 female Wistar rats were placed in time biology laboratory and, according to a fixed light(L): a dark(d) time(L: D = 12: 12, light period for 07: 00 to 19: 00 dark period for 19: 00 ~ 07: 00), free feeding drinking water. Rats were randomly divided into blank group?model group?TG group ?MTX group and LEF group. 8 rats were in blank group. 16 rats were in model group. 32 rats were in TG group. 32 rats were in MTX group. And 32 rats were in LEF group. Raised 10 days later, the rats were established of collagen induced arthritis(CIA) model. Then we should observe model rats and mark the rat ankle and foot swelling degree. TG(40mg/kg)?MTX(0.1mg/kg) and LEF(2mg/kg) were administered orally at ZT6 or ZT18 for 42 d. TG and LEF were administered every day. MTX was administered every two days. In 42 d, blood was taken at different time(ZT2,6,10,14,18,22 hour). 2 Experimental methods 2.1 Using vernier caliper measure ankle joint measurement of rat paw thickness and diameter every 3days. 2.2 The expression levels of TNF-, IL-6 and IL-17 a in rat serum were detected by ELISA. 2.3 The expression levels of, IL-17 a, MMP-3, TNF-?, BMAL1, CLOCK and IL-6 were detected by immunohistochemistry method. And the pathological morphology was observed under the microscope. 2.4 In 41 d the rats were shot X-ray films of double feet to observe the progress of imaging. 2.5 The normal distribution of measurement data are the mean standard deviation((?)±s) said. The variance analysis of multi sample mean was used to judge the difference between the two two groups, and the t test was used to judge the difference between the two groups. Test level ? =0.05. Results 1 Study on circadian rhythm of related inflammatory factors in CIA rats(Part one) 1.1 Comparison of joint swelling degree in each group of CIA ratsWith the prolongation of the time of CIA modeling, the degree of swelling of the joints increased first and then decreased slowly. In model group CIA rats, 33 days after first immunized, the ankle joint was the most serious. 39 days after first immunized, the swelling of the palm and finger is the most serious. 1.2 Contents of IL-17 a, IL-6 and TNF- in serum of TG6 h groupThe serum levels of IL-17 a, IL-6 and TNF- were different in each time point of CIA rat model. In one day, serum levels of IL-17 a, IL-6 and TNF-? were stable in the blank group; the serum level of IL-17 a, IL-6 and TNF-? were similar to each other during the day and night. The serum level of IL-17 a began to rise at ZT18, and reached the peak at ZT22; began to decline at ZT2, and reached the valley at ZT6. The serum level of IL-6 began to rise at ZT14 and peaked at ZT22; began to decline at ZT2, and got into the platform at ZT6. TNF-? began to rise at ZT14 and peaked at ZT22; began to decline at ZT2, and got into the valley at ZT6. 1.3 X-ray observation of ankle joint of CIA rats in each groupThe X-ray films of the ankle joint of rats in each group were different. In the blank group, the ankle joint X-ray was observed in the ankle joint. The joint surface of the ankle joint was clear, the joint of the palm and finger was complete, and the joint space was not abnormal. In the model group, the joint surface of CIA rats was vague, the bone defect was obvious, and the joint space was changed. 1.4 HE staining and immunohistochemical observation of CIA rats in each groupThrough HE staining, we observed that the synovium of rat ankle in Control group are smooth. There were 1 or 2 synovial cells without mastoid protrusion or hyperplasia. And we didn't observe infiltrating multinucleated cells. The synovial cells of CIA rat ankle were hyperplastic seriously in Model group, and there were mastoid bulge and infiltration of inflammatory cells. Through Immunohistochemical test, compared with Control group, the expression of IL-17 a, IL-6, TNF-?, MMP-3, CLOCK and BMAL1 were all increased in the rats ankle synovial cells of Model group. 2 Effect of tripterygium glycosides on the circadian rhythm of related inflammatory factor in CIA rat 2.1 Changes of the swelling value of CIA rats joint in each group After observation for 42 d, swelling level changes of CIA rats joint are not identical in each group. As the extension of building model time, the swelling level of CIA rats joint increased gradually. Compared with TG-ZT6 group, the swelling levels of ankle foot diameter and paw thickness were decreased significantly(P<0.05).The change curve of foot paw thickness in TG-ZT18 group was always located under TG-ZT6 group by a continuous observation. 2.2 Content of IL- 17 a, IL- 6 and TNF-? of CIA rats serum in each group After intervention for 42 d, the content of IL- 17 a, IL- 6 and TNF-? of CIA rats serum in each time-treated group were different. Compared with TG- ZT6 group, the content of IL-17 a, IL- 6 and TNF-? of CIA rats serum were significantly reduced in TG- ZT18 group(P < 0.05). 2.3 X-ray observation of CIA rats ankle in each group X-ray radiography results of rats ankle in the two groups are obviously different. In TG-ZT6 group, the ankle joints of CIA rats were destructed, the articular surfaces were undefined, and there were bone defect in partial joint. In TG- ZT18 group, the ankle joint surface of CIA rats can be seen, the change in left foot was light, there were bone destruction in a small number of metacarpophalangeal joints, and the gap of joint changed light. 2.4 HE staining and immunohistochemical observation of CIA rats ankle in each group The synovial cells of CIA rat ankle were hyperplastic in TG- ZT6 group, and there were mastoid bulge and infiltration of many a large number of inflammatory cells. The synovial cells of CIA rat ankle were hyperplastic slightly in TG – ZT18 group, and there were mastoid bulge and infiltration of a few inflammatory cells. Through Immunohistochemical test, compared with TG- ZT6 group, the expression of IL-17 a, IL-6, TNF-?, MMP- 3, CLOCK and BMAL1 were all increased in the rats ankle synovial cells of TG – ZT18 group. 3 The mechanism of time treatment of common anti-rheumatic drugs in CIA rats 3.1 Changes of the swelling degree of CIA rats joint in each group After observation for 42 d, swelling degree level changes of CIA rats joint are not identical in each group. Compared with the model group, MTX group, LEF group and TG group were significantly decreased(P< 0.05). The ankle swelling degree was the most obvious in MTX, and the thickness of foot claw was the most obvious in LEF group. The 42 days after administration MTX-ZT18 swelling group is significantly lower than MTX-ZT 6 groups(P<0.05). The 21 d after administration the swelling degree curve of MTX-ZT18 group was under MTX-ZT6. The 42 d after administration compared with TG-ZT 6 group swelling degree of TG-ZT18 groups decreased significantly(P<0.05). But TG-ZT6 group took effect slowly. The 42 d after administration LEF-ZT6 groups had no difference with LEF-ZT18 group(P>0.05). 3.2 Content of IL- 17 a, IL- 6 and TNF-? of CIA rats serum in each groupThe 42 d after intervention each treatment group CIA rats serum IL-17 a, IL-6 and TNFcontent was different. Compared with model group, the content of IL-17a?IL-6 and TNF-? of serum levels in two MTX treatment group were significantly decreased(P<0.05). Compared with the MTX-ZT6, the content of IL-17a?IL-6 and TNF-? of serum levels in two MTX-ZT18 group was significantly decreased(P<0.05). Compared with model group, the serum levels of IL-17 a, IL-6 and TNF-a levels in two LEF treatment group were significantly decreased(P<0.05). There was no difference in IL-17 a, IL-6 and TNF-a levels of LEF-ZT6 group and LEF-ZT18 group(P>0.05). Compared with the model group, IL-17 a, IL-6 and TNF-? serum levels of TG-ZT18 group decreased significantly(P<0.05). Compared with model group,the serum IL-6 and TNF-a levels of TG-ZT6 decreased significantly(P<0.05). But serum levels of IL-17 a had no difference with model group(P>0.05). Compared with group TG-ZT6, the serum IL-17 a, IL-6 and TNF-a levels of TGZT18 group significantly decreased(P<0.05). 3.3 X-ray observation of CIA rats ankle in each groupThe X-ray films of the ankle joint of rats in each group were different. In the normal group, the ankle joint X-ray was observed in the normal group. The joint surface of the ankle joint was clear. The joint of the palm and finger was complete. And the joint space was not abnormal. In model group, the CIA ankle joint was vague and the bone defect was obvious. The joint space was changed with the large bone defect of the palm and finger joint. MTX-ZT6 group CIA rats ankle joint was destructed. The joint surface was blur. And the partial palm finger joint visible bone was defected. MTX-ZT18 group CIA rat ankle joint surface is still visible. The left foot change is lighter. Less part of the finger joint bone was destructed. Slight changes was in the joint gap. LEF-ZT6 group and CIA group LEF-ZT18 rats ankle joint surface were visible. There was no obvious bone destruction. TG-ZT6 group CIA rat ankle joint were destructed. The joint surface was blur. Partial joint bone defect was visible. TG-ZT18 group CIA rat ankle joint surface was still visible. The left foot change is lighter. Less part of the finger joint bone was destructed. Slight changes was in the joint gap. 3.4 HE staining and immunohistochemical observation of CIA rats ankle in each groupThe blank group HE staining of rat ankle synovial was smoothly. 1-2 layers of synovial cells can be found. No papillary projection?hyperplasia and no more nuclear cell infiltration was fouond. In the ankle joint model group rats CIA HE staining we could see the proliferation of synovial cells, papillary bulge and a lot of inflammatory cells. TG group, LEF group and MTX group CIA rat ankle HE staining showed that synovial cells had different degree hyperplasia and inflammatory cell infiltration. TG-ZT18, LEF-ZT18, MTX-ZT18 group compared to TG-ZT6, LEF-ZT6 and MTX-ZT6 respectively,we could find inflammatory cells infiltration decreased of synovial cells. Immunohistochemistry observation of IL-17 a, IL-6, TNF-?, MMP-3, clock and BMAL1 expression levels of model group rats synovial cell were significantly higher than those in the blank group. compared with the model group, IL-17 a, IL-6, TNF-a, MMP-3, CLOCK and BMAL1 expression level of TG group,LEF group and MTX group CIA rat ankle joint synovial cell and is significantly reduced. compared with TG-ZT18, LEF-ZT18, MTX-ZT18 group respectively,the IL-6, TNF-a, MMP-3, CLOCK and BMAL1 expression of TG-ZT6, LEFZT6, MTX-ZT6 IL-17 a of synovial cells were decreased. Conclusions 1 The serum levels of IL-17 a, IL-6 and TNF-? in CIA rats were all reached the peak value of ZT22 and reached the valley at ZT6. It showed dynamic changes in the day and night.2 The administration of TG and MTX in the increasing of inflammation can inhibit the inflammation and slow down the disease more effectively.3 TG, LEF and MTX may affect the expression of the clock gene directly /indirectly and affect the level of inflammatory cytokines to make countribution to anti-inflammatory. |
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