Study On Related Molecular Mechanism Of Needle Knife On Knee Osteoarthritis

Objective:To evaluate the therapeutic effect of needle knife treatment by using rabbit KOA(Knee Osteoarthritis)model,to explore the target of needle knife release therapy,and to describe the molecular mechanism of needle knife release on knee osteoarthritis.To provide theoretical basis for clinical application of needle knife treatment.Methods:Using a 2%papain as a solubilizer,a multi-site articular cavity injection was performed on the left hind limbs of New Zealand rabbits on days 1,3 and 5 respectively,and the rabbit KOA model was constructed by combining the forced flexion of the joints for one week.To evaluate KOA models by X-ray film and lateral film combine with Lequesne MG knee level assessment.KOA rabbits that successfully constructed were randomly divided into model group,electroacupuncture control group,drug(sodium hyaluronate)control group,and needle knife group,and normal rabbits as blank control group.Each group was given a corresponding treatment intervention.The needle knife group was treated once a week,the electroacupuncture group was treated twice weekly and 20 minutes/time,the sodium hyaluronate group was twice weekly,and 0.4 ml/time.After 4 weeks of treatment,cartilage tissue was collected and relevant indicators were evaluated.(1)Evaluation of the knee joint behavioral changes by the Lequesne MG knee joint evaluation method.The radiographic changes of the knee joints were evaluated with an X-ray film of the left hindlimb,and quantitative comparisons were made according to the Kellgren-Lawrecne imaging grading standard.(2)The expression of biomarkers MMP-3 and inflammatory cytokines TLR4,NF-κB,IL-1β and TNF-α in knee cartilage were detected by ELISA.(3)The expression of MMP-3,IL-1β,TNF-α,TLR4 and NF-κB in knee cartilage was quantified by immunohistochemical staining,and MMP-3,IL-1β,TNF-α and TLR4,NF-κB expression levels were quantitatively analyzed by pathological image analysis system.(4)Real-time PCR was used to detect the mRNA expression of MMP-3,IL-1β,TNF-α,TLR4 and NF-κB in cartilage tissue.(5)The proteins contents of MMP-3,IL-1β,TNF-α,TLR4 and NF-κB were detected by Western blot in knee cartilage.Results:(1)The behavioral observations showed that the articular cartilage in the KOA model group had a dull appearance,rough surface,and even erosion.Some of the cartilage had ulcers,and most have osteophyte formation.The articular surface of the needle knife release treatment group was smooth and the joint edges were smooth,and rare osteophyte formation;Lequesne MG quantitative analysis showed that Lequesne MG score was significantly higher in KOA model group compared with the blank control group,the difference was significant(P<0.01).The treatment intervention group can significantly reduce this elevated effect,the difference was statistically significant(P<0.01),and the effect of needle knife group was significantly better than sodium hyaluronate and electroacupuncture positive control group(P<0.05).(2)Observed on X-ray film,the knee joints of KOA model group were swollen,the articular surface was rough,the degeneration was obvious,the osteophytes around the joints formed,the high-density shadows appeared in the subchondral bone,the joint space became narrow.In the needle knife group,the knee joint surface was relatively smooth,with a small number of visible joint space stenosis and high density of subchondral bone.Compared with the KOA model group,the joint degeneration of the needle knife group improved significantly.Kellgren-Lawrecne quantitative analysis showed that this improvement was significant(P<0.01).The improvement effect was consistent with the electroacupuncture positive control group,but the effect was better than the sodium hyaluronate positive group.(3)The results of ELISA showed that the levels of MMP-3,IL-1β,TNF-α,TLR4 and NF-κB in the KOA model group were significantly increased(P<0.01).Compared with the KOA model group,the therapeutic intervention group significantly reduced the above indicators.and the difference was significant(P<0.05,P<0.01);the effect of the needle knife release group on the above indicators was significantly better than sodium hyaluronate group and electroacupuncture positive control group(P<0.01).(4)Qualitative statistic analysis of immunohistochemical staining showed that the positive signal appeared brownish brown.In the KOA model group,MMP-3,TNF-α,IL-1β and TLR4,NF-κB immunohistochemical staining were positive,and the microscope showed brown-yellow heterogeneous particles.And brown-yellow heterogeneous particles on all treatment intervention group have a reduction in different degrees,needle knife release group has the least yellow heterogeneous particles.Image quantitative analysis showed that MMP-3,TNF-α,IL-1β and TLR4,NF-κB positive expression rate in KOA model group significantly increased(P<0.01),each treatment intervention group can obviously reduce the positive expression rate of the above factors,the difference was statistically significant(P<0.05,P<0.01).The reduction effect of the needle knife release group is the most significant,the effect is better than the sodium hyaluronate and electroacupuncture positive control group(P<0.01).(5)The results of Real-Time PCR and Western Blot showed that the mRNA and protein expression of MMP-3,TNF-α,IL-1β,TLR4 and NF-κB were significantly up-regulated in KOA model group,and the difference was significant(P<0.01).Each groups with treatment intervention down-regulated the expression of mRNA and protein above.And the down-regulated effect of needle knife group significantly better than sodium hyaluronate and electroacupuncture positive control group(P<0.05,P<0.01).Conclusions:(1),The typical extracellular matrix degrading enzyme MMP-3 is highly expression in the knee osteoarthritis model,and can be used as a biomarker for evaluating the efficacy of anti-knee osteoarthritis.Needle knife can significantly reduce the expression of MMP-3.(2)Needle knife has a definite therapeutic effect on knee osteoarthritis,and the therapeutic effect is better than electroacupuncture and sodium hyaluronate.(3)TNF-α,IL-1β and NF-κB inflammatory cytokines mediate the occurrence and development of knee osteoarthritis,and needle knife can significantly reduce this inflammatory expression to relieve inflammation symptoms of knee osteoarthritis.That is,the effect of needle knife on the treatment of knee osteoarthritis is closely related to the reduction of inflammatory cytokines TNF-α,IL-1β and NF-κB expression.(4)TLR4-NF-κB signaling pathway is involved in the pathogenesis of knee osteoarthritis.Needle knife can significantly reduce the expression of TLR4 and NF-κB pathway factors in TLR4-NF-κB signaling pathway.That is,the therapeutic effect of knee osteoarthritis is closely related to the activation of TLR4-NF-κB signaling pathway.(5)The molecular mechanism of needle knife on knee osteoarthritis is related to the regulation of TLR4-NF-κB signaling pathway,reduction the expression levels of the downstream inflammatory cytokines IL-1β,TNF-α,and downregulation activity of MMP-3.This leads to the reduction of degradation on the extracellular matrix.