| ObjectiveTo observe the therapeutic effect of RNA interference for Bcl-xl oncollagen-induced arthritis (RA) in rats, and apoptosis-related gene expression inRA synovial tissue, and to explore the pathogenesis of RA, in the hope of findingnew therapeutic targets.MethodsOf40pathogen-free SD rats,32were randomly selected to establish a ratmodel of collagen-induced rheumatoid arthritis (CIA). The remaining8wereused as normal control. CIA model was established with subcutaneouslymulti-sites local injection of0.8ml freshly prepared type II collagen emulsioninto the tails on the1st and7th day. On the9th day,24rats with arthritis indexgreater than2were randomly divided into3groups, namely the model group,sham treatment group and the treatment group. Treatment was given on the11th and13th, respectively.1ml saline was administered into the tails of rats inthe model group and the normal control group. Rats in the sham treatmentgroup received subcutaneous injection of15μl scramble siRNA dissolved in1ml saline in the rat tail vans, whereas rats in the treatment group were similarlytreated with15μl siRNA for Bcl-xl dissolved in1ml saline. Body weight, arthritis index and left hind toe volume were recorded every7days. Rats weresacrificed and their joint tissues obtained on the30th day of the experiment. HEsections were made to observe the pathological change in rat joints. Westernblotting was used to investigate the protein expressions of Bcl-xl, Bax,caspase-3in the synovial tissues. Bcl-xl and Bax mRNA expressions weredetected with RT-PCR method.Results1.The general situation of rat arthritis model: the body weight of rats in thenormal group continued to grow, the body weight of rats in the model group andthe sham treatment group continued to decline. Before the14th day of treatmentthe decrease was significant, but after that the decline was slow, and no growthwas observed. The body weight of rats in the treatment group decreasedsignificantly before the14th day, but gradually increased and showed nosignificant difference as compared with those in the model group and the shamtreatment group (P>0.05).2.Silencing Bcl-xl with RNA interference on the big norvegicu paw swellingdegree of the rat arthritis model: the degree of joint swelling increasedsingificantly during the experiment period in the model group and the shamtreatment group, which progressed to immobility at sacrifice. A few rats showedjoint deformity and skin ulcers. Rats in the treatment group developed obviousjoint swelling during the early stage of the treatment. After administration theswelling gradually eased, and did not show joint activity limitation beforesacrificed. The paw volume index was better than that in the model group andthe sham treatment group.3. The pathological changes of synovial tissue in the pathogenesis ofarthritis: there was only1-2layers of synovial lining in the synovial membrane inthe joints of the normal group and were not visible in some parts. No infiltrationof inflammatory cells in the synovial angiogenesis was found. The articular cartilage surface was smooth and free of damage or break. Synovial lining wasmarkedly thickened in the model group and the sham treatment group, withapproximately8-10layers in which cells were sparsely arranged. A largenumber of inflammatory cells infiltrated in the synovium, and in some partstypical pannus formation was visualized in the model group and negative controlgroup. Invasive growth of some pannuses was observedfrom the cartilage surface, causing the destruction of the cartilage surfaceand the bone tissue. Bony fusion was observed in a few of the joints undermicroscope. Treatment group compared with the control group showedpathological changes. But when compared with the model group and thenegative control group, the layer number of synovial lining was3-5fewer, withinfiltration of inflammatory cells in the synovial and no pannus formation. Thearticular cartilage surface was smooth and not significantly damaged.4. Influence of silencing Bcl-xl by RNA interference on Bcl-xl, Bax,Caspase-3protein expressions in synovial tissue: Westren blotting for proteinexpression showed that, compared with tissues from the normal group, Bcl-xlprotein expression level in the model group and the negative control group wassignificantly increased, and the difference was statistically significant (P <0.01).Significantly reduced protein expression of Bcl-xl in the treatment group wasdetected when compared with the model group and the negative control group(P <0.05), which was still higher than the normal group. Compared with thenormal group, protein expressions of Bax and Caspase-3within the synovialtissue in the model group and the negative control group were slightly increased,but the difference did not reach statistical significance (P>0.05). The treatmentgroup showed significantly higher expression levels as compared with thenormal group, the model group and the negative treatment group(P <0.05).5. Influence of silencing Bcl-xl by RNA interference on Bcl-xl and BaxmRNA expressions in synovial tissue: RT-PCR assay for Bcl-xl and Bax mRNA expressions in the synovial tissue showed that: compared with the normal group,Bcl-xl mRNA expression in the model group and the negative control group wassignificantly higher (P <0.01). When compared with the model group and thenegative control group, Bcl-xl mRNA expression in the treatment group wassignificantly lower (P <0.05). But when compared with the normal control group,the difference was not statistically significant (P>0.05). Compared with thenormal group, the expression of Bax mRNA was increased in the synovial tissueof the model group and the negative control group (P <0.05). When comparedwith the model group and the negative treatment group, Bax mRNA expressionwas significantly higher in the treatment group (P <0.05).Conclusions(1) The pathogenesis of rheumatoid arthritis was accompanied byabnormal expression of apoptotic regulators. Specifically there was asignificantly high level of expression of anti-apoptotic factors, causing animbalance between the proliferation and apoptosis of synovial cells, whichpromotes the development of RA.(2) RNA interference to silence Bcl-xl significantly down-regulated theexpression of anti-apoptotic factors promoted the expression of apoptoticfactors, thereby delaying disease progression of RA.(3) siRNA plasmid implanted via lent viral vector into rats in vivo couldexpress normally and act sustainably. |
PDF Full Text Request