| Objective The purpose of this study was to investigate the ameliorative effects of glucosamine(GS)and chondroitin sulphate(CS)on rheumatoid arthritis(RA)in rats and to compare the efficacy of the two functional foods alone and in combination,as well as to explore the mechanism of action of GS and CS in improving RA based on the perspective of the gut microbiota.Methods In this study,all SD rats were fed freely in an SPF-level experimental environment for two weeks,and the RA rat model was established by injecting Complete Freund’s adjuvant into the tail base of the rats.After the success of this model was determined using a specific toe swelling measurement instrument,the model rats were randomly divided into four groups: model blank group(MT group),glucosamine intervention treatment group(GS group),chondroitin sulphate intervention treatment group(CS group),and glucosamine combined with chondroitin sulphate intervention treatment group(GC group),and a blank control group(NT group),for a total of five groups.The day when the model succeeded or the intervention started was recorded as "D0" in the experiment.During the continuous intervention period,body weight and hindfoot toe volume were measured,and faecal specimens were collected from each group at different time points(D0,D4,D11,D18,D25,and D36).Serum and right ankle joint specimens were collected at the end of the experiment.TNF-α,IL-1,IL-10,PGE2,and MMP-3 levels in the blood were measured using enzyme-linked immunoassays,and NO levels were determined using the nitrate reductase method.HE staining was performed on the right ankle specimens and the expression of TLR-4 and NF-κB was determined by immunohistochemistry,followed by pathological changes observation and scoring.As determined by the above indicators,stool specimens from three time points(D18,D25,and D36)were subjected to 16 S r RNA gene sequencing as well as Spearman correlation analysis.Results The results of general and toe volume measurements revealed that the toe swelling in the model and intervention rats was statistically different from the normal group at about 14 days after adjuvant injection(P<0.01),with the peak of swelling occurring around 25 days after injection.After 25 days of continuous intervention with GS,CS and GC,GS and CS tended to alleviate toe swelling caused by the RA model,but there was no statistical difference,while GC significantly improved toe swelling caused by the RA model and was statistically different(P<0.05).In addition,the RA model causes histopathological damage to the toe,as evidenced by infiltration of inflammatory cells,formation of vascular opacities,severe synovial hyperplasia,and erosion of cartilage,etc.GS and CS tended to improve this histological damage without statistical differences,whereas GC significantly improved the pathological damage and was statistically different(P<0.05).Immunohistochemical results showed that TLR-4 and NF-κB were highly expressed in the ankle tissue of the RA model rats,while oral administration of GS,CS and GC lowered the expression levels of these two chemokines to some extent.Based on the 16 S r RNA gene sequencing results,we found that the RA model rats and the NT group were not statistically different in alpha diversity,but there were significant differences in the makeup of the gut microbiota(P=0.001).On the one hand,β-diversity analysis revealed a significant difference in the microbiota structure between the MT and NT groups of rats on the D18 and D25(P<0.05),while no statistically significant difference was seen in this microbiota structure on the D36(P=0.189).In addition,no significant difference was seen in the microbiota structure between the GS and MT groups,while in the CS group compared to the MT group,only a significant difference was found between the two at D25(P=0.002),while no other significant difference was seen.Notably,there was a significant difference in the microbiota structure between the GC and MT groups(P<0.05).On the other hand,we also found the following major genera enriched in the RA model,such as Escherichia_Shigella、Bilophila、Bacteroides、Jeotgalicoccus、Enterobacter、Nosocomiicoccus、NK4A214_group、Candidatus_Saccharimonas、Corynebacterium、Eggerthellaceeae_unclassfied、Lachnospiraceae_NK4A136_group and Clostidia_vadin BB60_group_norank.Combining the results of related studies and the correlation analysis in this experiment,we found that the above genera were dominated by inflammatory-promoting genera and lipopolysaccharide(LPS)-producing bacteria.Analysis comparing the GC and MT groups revealed that oral supplementation of GC decreased the abundance of Enterobacter and Erysipelotrichaceae,and it has been shown that these two genera are LPS-producing bacteria.GC also significantly increased the abundance of Lactobacillus,which was previously reported to be negatively correlated with LPS.In addition,Spearman’s correlation analysis showed a significant correlation between the gut microbiota and clinical phenotype,e.g.,Esherichia_Shigella and pro-inflammatory factor TNF-α content were positively correlated(P<0.01),while Candidatus_Sasccharimonas and Nosocomiicoccus were positively correlated with right hindfoot swelling(P<0.01).NK4A214_group and Corynebacterium were positively correlated with pathology scores(P<0.05),among others.Conclusions 1.Both GS,CS and GC can alleviate the symptoms of RA-related joint inflammation and swelling to some extent,but GC has a more pronounced impact.2.Previous studies have confirmed the improvement effect of GS and CS in osteoarthritis,and the results of this experiment show that these two functional foods also have a certain improvement effect in RA,expanding the application range of GS and CS.3.The microbiota diversity analysis showed that the RA model leads to the development of gut microbiota dysbiosis symptoms,and such symptoms can be better recovered after the intervention treatment,and the improvement effect of GC is superior to that of GS and CS.4.Analysis of the 16 S r RNA gene sequencing results showed that the main genera enriched in the RA model,Escherichia_Shigella,Bilophila,Bacteroides,Jeotgalicoccus,Enterobacter,Nosocomiicoccus,NK4A214_group,Candidatus_Saccharimonas,Corynebacterium,Eggerthellaceeae_unclassfied,Lachnospiraceae_NK4A136_group,and Clostidia_vadin BB60_group_norank,combining the results of previous reports and this experiment revealed that the above genera were mainly associated with inflammation.5.Spearman correlation analysis linked changes in the gut microbiota to clinical phenotypes and showed that most of the genera enriched in the RA model were positively correlated with the expression levels of cytokines that promote inflammation,hindfoot swelling and pathology scores,while negatively correlated with anti-inflammatory factors.6.The results showed that the gut microbiota was involved in the improvement of RA by GC,probably through the following pathways: GC influenced the production of LPS by regulating LPS-related genera,which in turn inhibits the activation of the TLR-4/NF-κB pathway,alleviating RA-induced joint inflammation and improving the manifestation of joint swelling and injury.7.Previous studies have confirmed that the symptoms of intestinal dysbiosis appear earlier than the symptoms of RA,and the results of this study further suggest that the improvement of dysbiosis symptoms precedes the improvement of clinical symptoms in the process of improving RA by GC. |
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