基于细胞外信号调节激酶通路探讨筋滞骨错手法对兔膝骨关节炎软骨退变的影响及作用机制OA

Objective To investigate the effects of the sinew stagnation and bone dislocation manipulation on cartilage degen-eration in rabbits with knee osteoarthritis(KOA)via the extracellular signal-regulated kinase(ERK)pathway and to elucidate its underlying mechanisms.Methods A total of 30 healthy male New Zealand White rabbits were randomly divided into blank group(n=10),and modeling group(n=20).The blank group did not received treatment,whereas the modeling group adopted the Videman modeling method to establish an early KOA model.Due to modeling failure and death during the intervention period,8 and 16 cases were ultimately included in the blank group and the modeling group,respectively.The modeling group was further divided into model group and manipulation group,with eight cases in each group.The blank group and the model group were permitted free activity and diet without any intervention.The manipulation group received sinew stagnation and bone dislocation manipulation technique after successful modeling,including manipulation of regulating tendon stagnation(manipulation along the three Yang meridians of the foot and three Yin meridians of the foot;pressing acupoints such as Futu(ST32),Liangqiu(ST34),medial and lateral Xiyan(EX-LE4,EX-LE5),Xuehai(SP10),Yanglingquan(G34),Ashi acupoints,at a rate of 60-80 times per minute,for two minutes per acu-point,12 minutes per session,once a day,for a total of 21 days,and the bone dislocation correcting technique once a week,three times a course,with a total intervention of 21 days.The Lequesne Algofunctional Index(Lequesne MG score)was used to assess rabbit behavior and gait.Hematoxylin-Eosin(HE)staining was used to observe pathological changes in rabbit knee joints.Safranin O-Fast Green staining was used to evaluate structural alterations in articular cartilage and subchondral bone.Western blot analysis was carried out to determine the protein expression levels of ERK1/2,phosphorylated ERK1/2(pERK1/2),matrix metalloproteinase-13(MMP-13),and tumor necrosis factor-alpha(TNF-α).Quantitative reverse transcription polymerase chain reaction(qRT-PCR)was applied to measure the mRNA transcription levels of ERK1/2,MMP13,interleukin-1 beta(IL-1β),and TNF-α.Micro-CT was utilized to assess trabecular connectivity density(Conn.D)and trabecular number(Tb.N)in rabbit knee joints.Results(1)Lequesne MG score:compared with the blank group,the Lequesne MG scores of the model group and the manipulation group increased signifi-cantly after intervention(P<0.05).Compared with the model group,the Lequesne MG score in the manipulation group decreased sig-nificantly after intervention(P<0.05).(2)Cartilage pathological changes:the cartilage surface of the medial plateau was uneven in the model group,with local loss of cartilage.The superficial chondrocytes showed a decrease in number and hypertrophic differentia-tion,while the deep chondrocytes were clustered.Obvious cartilage fissures or defects were observed,and cartilage matrix staining was uneven.The superficial cartilage in the manipulation group was more regular compared to that of the model group.The arrange-ment and distribution of chondrocytes were more uniform,and cartilage matrix staining was more even.(3)Expression levels of TNF-α,MMP-13,ERK1/2 protein and pERK1/2 proportion:compared with the blank group,the expression levels of TNF-α,MMP-13,ERK1/2 protein,as well as the proportion of pERK1/2 in the model group increased significantly after intervention(P<0.05).Compared with the model group,the expression levels of TNF-α,MMP-13,ERK1/2 protein,as well as the proportion of pERK1/2 in the manipulation group decreased significantly after intervention(P<0.05).(4)The mRNA transcription level of MMP-13,IL-1β,TNF-α,and ERK1/2:compared with the blank group,the mRNA transcription level of TNF-α,MMP13,and IL-1β in the model group increased significantly after intervention(P<0.05).Compared with the model group,mRNA transcription levels of MMP-13,IL-1β,TNF-α,and ERK1/2 in the manipulation group decreased significantly after intervention(P<0.05).(5)Bone mineral density and trabecular number:compared with the blank group,the values of Conn.D and Tb.N in the model group decreased significantly after intervention(P<0.05);the trabecular structure was irregular,and the osteophyte formation increased significantly.Compared with the model group,the values of Conn.D and Tb.N in the manipulation group increased significantly after intervention(P<0.05),the trabecular structure was more regular,and the osteophyte formation decreased significantly.Conclusion The sinew stagnation and bone dislocation manipulation technique can improve the knee joint structure and knee joint function of KOA model rabbits,and its mechanism may be related to regulating the ERK pathway to inhibit inflammatory responses and extracellular matrix degradation,and alleviate endoplasmic reticulum stress damage.