| Osteoarthritis (OA)，one of the most common chronic degenerative joint lesionsfor mammals, can be considered to be part of the aging process. It is characterized bythe progressive pain, stiffness, hypertrophy and the movement restriction of thedamaged knee, which has been attracted great interest in this field. The incidence ofOA increases with age, of which the tendency would be markedly visible as China isbecoming an aging society. Although the exactly mechanism of the disease isunclear the pathogenesis of OA is characterized by the degeneration of articularchondrocytes. The clinical work is lack of effective treatment except forsymptomatic treatment. Relieve symptoms and a few cases do prostheticreplacement of joint. This situation has become the bottleneck of restricting theclinical treatment of OA and the development of an ideal drug target to OA.Therefore, strengthening the research in the etiologic and biological field of OA andfinding a new target for the effective treatment of OA can not only improve thequality of life for elderly person, but also reduce the economic burden for the familyand society. In all, it is significant for improving the level of national health.The knee OA is the most common among the whole OA disease, the symptomsof which includes the pain and limp of the knee (especially when going up and downstairs that needs joint activities) in the early phase and the moving restriction of thejoint, muscle atrophy and the knee deformity in the late phase. Currently, most of theresearches in the field of the pathogenesis of knee OA are centering on the variability and degradation of the articular cartilage cells and their extracellular matrix(collagen and proteoglycan) and the roles of inflammatory cytokines involving it.Specifically, the roles of the inflammatory cytokines in the development of OA haveattracted the most attention in the recent decade. It is expected that the biologicalmeasures can be utilized in the effective treatment of OA through regulating theinflammatory cytokines in the OA pathogenesis,In the end of1980s, series cross-sectional epidemiological studies and cohortstudies by Felson and his colleagues revealed that smoking negatively correlatedwith osteoarthritis. In their studies, he firstly evaluated the relationship between OAprevalence and smoking in1415subjects during1983-1985and founded thatcompared to non-smokers (37.5%), the rate of OA was significant lower in smokers(28%). Data analyses have shown consistently that smoking modestly protect againstknee OA (relative rick (RR)=0.74,95%CI [0.57-0.95]), especially for severe OA(RR=0.70,95%CI [0.94-0.98]). Subsequently, Felson et al. performed a longitudinalstudy of knee OA to the subjects. Weight bearing knee radiographs were obtained in1983-1985(baseline) and again in1992-1993. Data analyses have shown smokershad a lower rate than did non-smokers (OR=0.4,95%CI [0.2-0.8]), which indicatedthat smoking would well have a protective effect against OA, but the mechanismremains unclear.Nicotinamide, commonly known as nicotine, is the main component of tobacco,which belongs to Pyridine family of alkaloids, is the agonist of nicotinicacetylcholine receptor (nAChR). The latest research has demonstrated that nicotinecould activate nAChR to exert regulation in the aspect of immune inflammatorythrough cholinergic anti-inflammatory pathways, which is also considered as one ofthe most important immune anti-inflammatory mechanism in the body. A large number of studies have shown that the cholinergic anti-inflammatorypathway has closely relationship to the pathological process in various disease suchas sepsis, Alzheimer’s disease (AD), epilepsy, schizophrenia, lung cancer, type2diabetes mellitus (DM), myasthenia gravis (MG), Parkinson’s disease (PD), vasculartumors, inflammation, ischemia reperfusion injury, rheumatoid arthritis and otherautoimmune diseases. The cholinergic anti-inflammatory pathway is an importantdirection of inflammation intervention as it can act on specific tissue to inhibitmultiple inflammatory mediators directly and rapidly.The first part of this work utilized rat knee OA model to study the dynamicalterations of nAChRs (especially the main subunits such as α7, α4, and β2) in thearticular cartilage of OA model and then to clarify the relationship between nAChRsand the occurrence and development of OA. The second part would continue toobserve the protective effect of nicotine on the cartilage of rat knee osteoarthritisbased on the MIA rat OA model.Part1Establishment of rat osteoarthritis model and thealterations of gene expression of nicotinic acetylcholine receptorin articular cartilageObjects: To establish a monoiodoacetic acid (MIA)-lesioned animal model ofOA in rats so as to observe dynamic alterations in the gene expression of the α7, α4and β2subunits of nicotinic acetylcholine receptor (nAChRs) in articular cartilage.Methods: Twenty adult SD rats were classified into control group and MIAgroup randomly. The right knee of the rats in MIA group received the intra-articularinjections of MIA (1mg,50μl) weekly sustained for4weeks. On the other hand, the rats in the control group would receive a intra-articular injections of saline weekly inthe right knee last for4weeks. All the left knees of rats were without surgery forown-control design. Rats in both groups were killed on the15thand30thday aftersurgery separately so that half for each time. The right knee samples were carefullydissected and observed the destruction of articular cartilage by generally lightmicroscope and histological pathological examination. In addition,reverse-transcription PCR was utilized to detect the gene expression of type Ⅱcollagen and aggrecan and the real-time PCR were used to evaluate the alterations ingene expression of nAChRs α7, α4and β2in articular cartilage.Results: The rats in both groups were all recovered after surgery.1) comparedto the control group, the light microscopy score and the Mankin’s score in MIAgroup were significantly higher, which suggested the MIA group appearedsignificant histological changes after MIA treatment for15or30days.2) The geneexpression of type Ⅱ collagen and aggrecan of the right kneesin the MIA groupwere remarkably lower than the control group in a time-dependent manner.3) Thegene expression of nAChRs α7, α4and β2of right articular cartilage in the MIAgroup had significantly increased on Day30compare to control group.Conclusion: The intra-articular injections of MIA can significantly destruct therat knee joint cartilage. The dynamic alterations in the gene expression of nAChRsα7, α4and β2subunits in articular cartilage can lay the experimental foundation forstudying the relationship between the signaling pathway of choline and OApathological mechanisms. PART2The protective effect of nicotine on the osteoarthritiscartilage in ratObjects: To study the protective effect of nicotine intervention on the cartilagein the rat OA model.Methods: Forty adult SD rat were classified into four group randomly, which iscomposed of Non-operation group (Con group), operation group (MIA group), andtwo nicotine intervention group including MIA+Nic0.25mg kg-1(N1group) andMIA+Nic0.5mg kg-1(N2group). The rats in N1and N2group received continuousintraperitoneal injection of nicotine as the pre-treatment for one week once a daybefore surgery. Simultaneously, the rats in both control group and MIA group didcontinuous intraperitoneal injection of saline as the pre-treatment. Afterpre-treatment, the right knee of the rats in MIA group, N1group and N2groupreceived the intra-articular injection of MIA (1mg,50μl) weekly for4weeks, whilethe rats in control group received intra-articular injection of saline. All the left kneesof rats were without surgery for own-control design. Additionally, the rats in N1group and N2group received intraperitoneal injection of nicotine (0.25mg kg-1or0.5mg kg-1) as the intervention. Rats in all groups were killed on the15thand30thday after surgery separately so that half for each time. The right knee samples werecarefully dissected and observed the destruction of articular cartilage by generallylight microscope and histological pathological examination.. Reverse-transcriptionPCR was used to detect the gene expression of type Ⅱ collagen and aggrecan.Results: all rats in each group were recovered after surgery.1) Compared to thecontrol group, the light microscopy score and the Mankin’s score in MIA group weresignificantly higher, which suggested the MIA group appeared significant histological changes after MIA treatment for15or30days2) Despite the geneexpression of type Ⅱcollagen and aggrecan of the right knees of the MIA groupwere remarkably lower than the control group, that of N1and N2group hadsignificant increased compare to MIA group in a does-dependent manner.Conclusion: nicotine has a protective effect on the knee cartilage of OA in a ratmodel.In summary, the main innovation of this work lies in:We established the OA rat model through inter-articular injection and indicatedthat nicotine has protective effect on articular cartilage in the rat OA model, themechanism of which was closely to nAChRs. This provides a new approach for theeffective treatment of OA and a novel theoretical basis for anti-inflammatoryprotective agent target to nAChRs. |
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