Effects And Mechanisms Of Vitamins On Serum Leptin Levels And Other Related Cytokines In Mice With Rheumatoid Arthritis

Background and objectiveRheumatoid arthritis(RA) is an autoimmune inflammatory disease characterized by proliferative synovitis which has been shown to be associated with the destruction of articular cartilage and deprivation of joint’s function.RA is a common disease worldwide.Epidemiological investigations revealed the incidences of RA in different regions,for example, the incidence in Chinese is0.32～0.36%,while in European adults,the incidence ranged from0.2～0.8%. People in Southern European regions including Italy and Greece had a low incidence of0.2～0.4%. Reaseachers had found that the incidence of RA is increased with the growth of age which culminated in people over65or70years.Females are more susceptible to RA than males.The mechanism of RA is complex involving heredity、nutrition、infection et al which has not been fully elucidated. Plenty of experiments showed that a complex cytokine networks exsited in RA, the biological effects of which is associated with the relative serum concentrations of inflammatory cytokines and their inhibitors.The interactions of cytokines played an important role in the inflammation、adhesion、 neovascularization and decreased bone density.Leptin is a type of hormone secreted by adipose tissue with a molecular weight of16000.It has long been regarded with relation to feeding and energy storage.It has been shown that leptin may act as a bridge between nutrition and immunology.Leptin has been indentified as an important cytokine in the inflammatory networks of RA. JAK/STAT signaling transduction pathway is a major way of leptin’s biological effects such as cell proliferation and differentiation、 immunological regulation and inflammation.Once leptin is combined to leptin receptor,corresponding moleculars are activated and transported to cell nucleus which promote the transcribing of target genes involving two key moleculars:STAT1and STAT3.RA is a chronic disease which require the intake of drugs including untirheumatics、NAIDs and biological agents. Patients are prone to drop out due to the side effects. It has been reported that33to75%of RA patients believe food plays an important role in their symptom’severity and20to50%will have tried dietary manipulation in an attempt to relieve their suffering. Anti-oxidants such as VitA and VitE manifested the inhibitory effects on inflammatory cytokines in vivo. It’s indicated vitamins with anti-oxidation property may be curable to RA.Therefore,our study aimed to observe the role of leptin in CIA mice, to examine the effect of VitA、VitE on leptin and other related experimental and clinical index and to explore the mechanisms based on JAK/STAT signaling transduction pathway.Materials and methodsAnimals and treatmentsMale Wistar mice weighed (147±15g) were used in the experiments. Both the animal care and study protocol employed were in accordance with Institutional Animal Care and Use Committee (IACUC) and the Organization for Economic Cooperation and Development (OECD) guidelines. The mice were housed in cages in a climate-controlled room with a12-hr light-dark cycle. Throughout the study, animals were fed with regular standard mice chow and water ad libitum. After1-week acclimation period, animals were given an intradermal injection of bovine type II collagen emulsified in incomplete Freund’s adjuvant or0.9%normal saline(the model and control groups, respectively). Two weeks later, the mice were given a booster intradermal injection. At the end of the4th week, arthritis index were applied to evaluate the paw swelling. Each paw was graded with a maximum score of4:0, normal,without any macroscopic signs of arthritis;1, mild, but definite redness and swelling of the ankle, or apparent redness and swelling limited to individual digits, regardless of the number of affected digits;2, moderate redness and swelling of the ankle;3, redness and swelling of the entire paw including digits;4, maximally inflamed limb with involvement of multiple joints. The4paw scores for each mouse were summed.Mice with a score higher than6were kept.Model group was divided into five subgroups:1) model group (n=6);2) VitA (42.86μgRE/kg.bw) group (n=6);3) VitE (200mg/kg.bw group (n=6); and4) Ibuprofen (50mg/kg.bw) group (n=6). Mice in the VitA、VitE and Ibuprofen groups received an intragastric administration of VitA、VitE、Ibuprofen respectively. At the end of the8th week, all the mice were anaesthetized and sacrificed, and blood samples and joint synovium tissue were extracted.The tissue was stored at-80℃until it was used for western blotting.1.Determination the level of serum leptin and other related experimental and clinical index.Blood samples were isolated by centrifugation at1000r/min for10min and then kept at-20℃before the following assay. The levels of leptin、TNF-α、IL-6、IL-10、 IL-4、CRP、RF were measured by ELISA using commercial kits.2.Western blot for STAT1and STAT3expression.Tissue samples were homogenized in complete RIPA lysis buffer. Total protein was quantified with the BCA protein assay kit. All preparations were carried out at 4℃. For western blotting, A Total of15μl of the mixture of protein and sample buffer was loaded per lane and electrophoretically separated on an SDS-PAGE gel. Protein bands were transferred to a PVDF membrane using a Trans-Blot SD Semi-Dry Electrophoresis Transfer Cell.The gels were then incubated with a primary antibody for p-STAT1and p-STAT3overnight at4℃. Finally, gels were incubated in secondary antibodies for1hr at room temperature.Statistical analysisData are expressed as means±SD. Statistical difference between two groups were measured by t test of two independent-samples.Statistical differences between groups were compared by one-way analysis of variance (ANOVA) with SPSS16.0software for statistical analysis. P<0.05was considered statistically significant.Results1.The induction of collagen-induced arthritis(CIA)Measured by arthritis index, mice with a score higer than6were regarded as a CIA model which is parallel with the pathogenesis of rheumatoid arthritis in human.2. Measurement on the levels of serum leptin、TNF-α、IL-6、IL-10、IL-4and ESR、 CRP、RFAs shown in Fig2,compared with the control,the serum leptin levels were significantly increased in model animals(P=0.000); Fig3showed the alterations in CIA mice assessed by serum TNF-α、IL-6、IL-4、IL-10levels.There was a significant increase in serum TNF-α、IL-6levels compared with control group(P=0.000);The level of serum IL-4、IL-10was significantly reduced compare with the control group(P=0.000); ESR、CRP and RF were makers of disease activity index in RA, Fig4-6. showed the changes.Compared with control group,the level of ESR、CRP and RF was significantly increased(P=0.000).3. The effects of VitA and VitE on arthritis index and the levels of serum leptin、 TNF-α、IL-6、IL-10、IL-4and ESR、CRP、RFFig7.showed the effect of VitA、VitE on arthritis index.Four-week administration of VitA and VitE significantly decreased arthritis index(P=0.008,0.014);As shown in Fig(8-12), the serum leptin、TNF-α、IL-6levels were significantly reduced in VitA and VitE group compared to model groupfor VitA group; the level of IL-10was significantly increased compared with model group(P=0.006,0.001); Fig(13-15). showed the effects of VitA, VitE on ESR、CRP and RF.Compared with model group,VitA、VitE can reduce the level of ESR and CRP significantly (P=0.025,0.021for VitA group,P=0.001,0.017for VitE group).4The effects of VitA、VitE on p-STAT1and p-STAT3protein expression levelp-STAT1and p-STAT3are the molecules of JAK/STAT signaling transduction pathway.Fig(16-17) showed the effects of VitA、VitE on p-STATl and p-STAT3protein expression level.The model group showed a significant up-regulation of p-STAT1and p-STAT3protein expression compared to model group(P=0.000).Mice treated with VitA、VitE for4weeks showed a a significant reduction of p-STAT3protein expression level(P=0.000).ConclusionOur study suggests the role of leptin on the pathogenesis of collagen-induced arthritis mice and the effects of VitA、VitE on cytokine network and other related index, the study also examined the effects of VitA、VitE on p-STAT1and p-STAT3protein expression level,the marker of lepin’s signaling transduction pathway. An unbalanced cytokine network exsisted in the pathogenensis of RA which manifested increased inflammatory cytokines and decreased anti-inflammatory cytokines. Leptin played an important role in RA and can be indentified as a inflammatory cytokine. Compared with model group,VitA、VitE can reduce the level of inflammatory cytokines such as leptin、TNF-α、IL-6and improve the level of anti-inflammatory cytokine such as IL-10. VitA、VitE can also decrease arthritis index and the level of ESR、CRP in CIA mice.There were reduced p-STAT1and p-STAT3protein expression levels in VitA and VitE group.The present study may provide a new way for curing RA.