| Objective: The study was carried out to investigate the influence of acupuncture and moxibustion on the cartilage tissue of OA model rats from liver-based therapy theory,and preliminarily elucidate the mechanisms of the circadian rhythm and acupuncture effects on cartilage injury,thus providing a scientific experimental basis for the prevention and treatment of OA from liver-based therapy theory.Methods: experiment 1 Thirty 12-week-old sprague-dawley male rats were choosed and divided into three groups,the model group,the sham operation group and the normal group randomly,10 for each group.The OA model rats were obtained using the modified Hulth method described as following.The anterointernal area of the right knee skin was cut and dissected carefully to expose the medial femoral condyle and the joint capsule in the model and sham operation grouos.Then the medial collateral ligament and the anterior cruciate ligament were cut off combined with the removing of the medial meniscus in rats of the OA model group.Rats In the sham group,only the medial femoral condyle and joint capsule were cut open and exposed.Rats in the normal groups were raised without any treatments.One week after the operation,the rats were put into the treadmill exercise device to run for 30 minutes a day for 3 weeks to replicate the OA model.Open field test,plantar pain threshold test,knee joint range of motion and other behavioral tests were carried out every week after modeling.Right knee joint imaging was checked.The ELISA test was used to measure the content of TNF-? and IL-1?in the synovial fluid.The damage of cartilage was measured by HE staining and Manikin's score.The content of Bmal1 and Clock protein in cartilage tissue was detected by immunofluorescence and western blotting.Experiment 2 Forty male SD rats of 12-week-old were choosed and divided into four groups randomly: the normal group,the OA model group,the acupoint selection from liver theory group,and the routine acupoint selection group,10 in each group.Rats in the normal group were raised without treatments,and the other groups were modeled.The treatment started 4 weeks after the model-making operation(that is,the treatment started after the end of 3 weeks of treadmill),and the treatment was 4 weeks in total.In the routine acupoint group,the Neixiyan,Waixiyan,and Zusanli were selected.In the acupoint selection from liver theory group,Yanglingquan,Ganshu,and Taichong were selected.Open field experiment,plantar pain threshold test,knee joint range of motion and other behavioral tests were carried out every week after modeling.The content of IL-1? and TNF-? in synovial fluid were detected by immunoenzyme-linked ELISA.HE staining of joint specimens and Mankin's score were used to observe cartilage damage,and the content of Bmal1 and Clock protein in cartilage tissue was detected by immunofluorescence and western blotting.Experiment 3 Fifty SD male rats of 12-week-old were choosed in this study and divided into 5 groups using the random number table method.Each group was treated as follows: 1)Normal group: no treatment.2)Model group: only model building without any treatment.3)ERK inhibitor group: model was made,intraperitoneal injection of ERK inhibitor 4 weeks after the modeling operation,continuous injection for 28 days.4)Acupoint selection from liver theory group: model was made,acupuncture treatment was started 4 weeks after model surgery,Ganshu,Taichong,and Yanglingquan were selected from liver theory.5)Acupoint selection from liver theory + ERK inhibitor group: model was made,intraperitoneal injection of ERK inhibitor 4 weeks after model surgery,continuous injection for 28 days,and acupuncture treatment from liver theory at the same time,select Ganshu,Taichong and Yanglingquan.The HE staining method and the Mankin's score were chosen to measure the damage of cartilage.The IL-1? and TNF-?contents in the articular fluid were measure by ELISA method.Immunofluorescence and Western blot were chosen to detect the protein contents of BMAL1,Clock,p-ERK,IL-1? and TNF-? in the cartilage tissue.Results: Experiment 1 1.After one week of operation,the total movement speed and distance,the mechanical pain threshold,thermal pain threshold,joint range of motion of the model group were reduced,and the rest time was prolonged in the OA model group when compared with rats in the normal group.Rats in the sham operation group only decreased in movement speed,the mechanical pain threshold and the thermal pain threshold.The total distance of movement,rest time,and joint range of motion were not significantly different comparing to rats in the OA model group.However,when compared to the normal group,the total movement distance,movement speed,and joint range of motion in the sham operation group increased,and the rest time was reduced;After 2-4 weeks of operation,the total movement distance and speed,the mechanical pain threshold,the thermal pain threshold and the joint range of motion decreased,and rest time prolonged,while the difference in open field test,the mechanical pain threshold,the thermal pain threshold,and joint range of motion in the sham operation group was not statistically significant(P>0.05).2.The imaging examination of rat knee arthritis showed that,in the normal group and the sham-operated group: there were joint gaps in the right knee joint,and the medial and lateral gaps were uniform,without obvious osteophytes and bone damage.Model group: The joint space of the right knee joint is narrowed,and the inner space is smaller than the outer side,and there is obvious osteophyte formation.3.The HE staining of cartilage specimens showed that,the Markin scores of the OA model group were significantly increased when compared to the scores of the normal group(P<0.01).However,there are no significant differences in terms of the Markin scores between the sham operation and normal groups(P>0.05).4.The immunoenzyme-linked ELISA method was used to detect the synovial fluid of the right knee in rats.The results show that,the content differences of IL-1? and TNF-? were not significant in the sham operation group when compared to the normal group(P>0.05).While the secretion of IL-1 and TNF-?increased significantly in the OA model group(P<0.01).5.Immunofluorescence and immunoblotting methods showed that, the expression level of proteins BMAL1 and Clock were down-regulated significantly in the OA model group compare to the normal group(P<0.01).While the protein expression differences were not shown between the sham acupuncture group and the normal group(P>0.05).Experiment 2 1.One week after the operation of the rats,compared with the normal group,there were significant differences in the open field test,thermal pain threshold,mechanical pain threshold,and joint range of motion in other groups(P<0.05),but there was no statistical significance among the modeling groups;After 2-4 weeks of the operation,the total distance of movement,the movement speed,thermal pain threshold,mechanical pain threshold,and joint range of motion in the other groups were significantly reduced comparing to the normal group,the rest time was significantly prolonged(P<0.05).However,no significant difference was detected between modeling groups.;At 5-8 weeks after operation,the rats were compared with the normal group,the total distance of movement,speed,thermal pain threshold,mechanical pain threshold,joint range of motion were all reduced significantly in different groups,and the prolonged rest time was shown in the result(P<0.05).When compared to the model group,the acupoint selection from liver theory group,and the routine acupoint group can increase the total distance of activity,thermal pain threshold,mechanical pain threshold,joint range of motion,and reduce rest time(P<0.05),From the 6th week,the routine acupoint selection group and the acupoint selection from liver theory group can improve the movement speed of rats(P < 0.05).Compared with the routine acupoint group,the acupoint selection from liver theory group can obviously increase the total distance of activity,thermal pain threshold,mechanical pain threshold,joint range of motion, and reduced rest time(P<0.05),and starting from the 7th week,the acupoint selection group from the liver therapy group can significantly improve the activity speed of rats(P<0.05).2.The HE staining of cartilage specimens showed that,compared with the normal group,the Markin scores of the other groups were significantly higher(P<0.001);the Markin scores results demonstrate a significant decrease in the routine acupoint group and the acupoint selection from liver theory group when compared to the OA model group.Besides,the Markin scores were decreased significantly in the acupoint selection from liver theory group comparing to the routine acupoint group(P<0.05).3.The use of immunoenzyme-linked ELISA to detect the synovial fluid of the right knee in rats showed an increasing level of IL-1? and TNF-?in the other groups comparing to the normal group(P<0.01).While a significantly decreased level of IL-1? and TNF-?was demonstrated in the routine acupoint group and the acupoint selection from liver theory group comparing to that of the OA model group(P<0.05).Compared with the routine acupoint group,the contents of IL-1? and TNF-? in the acupoint selection from liver theory group were less(P<0.05).4.Immunofluorescence and immunoblotting methods showed that,compared with the normal group,the contents of Bmal1 and Clock protein in the other groups decreased(P<0.01)while the protein expression levels of Bmal1 and Clock were significantly up-regulated in the treatment groups when compared to the OA model group(P<0.01).Moreover,the expression level of Bmal1 and Clock were all up-regulated in the acupoint selection from liver theory group when compared to the routine acupoint group(P<0.05). Experiment 3 1.The HE staining of cartilage specimens showed a significant increase of the Markin scores in other groups when compared to the normal group(P<0.001).While the decreasing scores were shown in the ERK inhibitor group,the acupoint selection from liver theory group and the acupoint selection from liver theory + ERK inhibitor group comparing to the OA model group(P<0.01);compared with the ERK inhibitor group,the Markin scores was not statistically significant difference in the acupoint selection from liver theory group(P>0.05),but the Markin scores were significantly reduced in the acupoint selection from liver theory + ERK inhibitor group(P<0.05);Compared with the acupoint selection from liver theory group,the Markin scores were significantly reduced in the acupoint selection from liver theory + ERK inhibitor group(p<0.05).2.The use of immunoenzyme-linked ELISA to detect the synovial fluid of the right knee in rats showed a significantly increased content of IL-1? and TNF-? in other groups compare to the normal group(P<0.01).While a decreased level was shown in the ERK inhibitor group,the acupoint selection from liver theory group and the acupoint selection from liver theory + ERK inhibitor group comparing to the model group(P<0.01);compared with the ERK inhibitor group,the content of IL-1? and TNF-? were not statistically significant difference in the acupoint selection from liver theory group(P>0.05),but the content of IL-1? and TNF-? were lower in the acupoint selection from liver theory + ERK inhibitor group(P<0.05);compared with the acupoint selection from liver theory group,the secretion of IL-1? and TNF-? were less than the acupoint selection from liver theory + ERK inhibitor group(P<0.05).3.Immunofluorescence and immunoblotting methods in the detection of rat articular cartilage tissue showed that,comparing to the normal group,the Bmal1 and Clock protein contents of the other groups decreased(P<0.05),and the P-ERK,TNF-? and IL-1? protein expression were up-regulated in other groups(P<0.05).When compared to the OA model group,the expression levels of the Bmal1 and Clock protein in the ERK inhibitor group,the acupoint selection from liver theory group and the acupoint selection from liver theory + ERK inhibitor group were significantly increased(P<0.01),the protein content of P-ERK,IL-1?,and TNF-? were significantly reduced(P<0.01);Compared with the ERK inhibitor group,the expression of Bmal1,Clock,P-ERK,IL-1?,and TNF-? protein in the acupoint selection from liver theory group were not statistically significant different(P>0.05);Compared with the ERK inhibitor group,the expression of Bmal1 and Clock protein in the acupoint selection from liver theory + ERK inhibitor group were significantly increased(P<0.05),and the protein content of P-ERK,IL-1?,and TNF-? were significantly reduced(P<0.05);compared with the acupoint selection from liver theory group,the content of Bmal1 and Clock protein in the acupoint selection from liver theory + ERK inhibitor group was higher(P<0.05),and the protein content of P-ERK,IL-1?,and TNF-? were significantly reduced(P<0.05).Conclusion: 1.The modified Hulth method can effectively replicate the OA model,and the circadian rhythm proteins of the model were abnormal.2.The effect of acupuncture and moxibustion selection from liver theory on the treatment of OA is better than routine acupuncture.3.Acupuncture and moxibustion selection from liver theory can improve joint function and repair damaged cartilage,which may be attributed to the decreasing of the ERK1/2 phosphorylation caused by the circadian protein,reducing the production of inflammatory substances,and reducing the destruction of cartilage matrix. |
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