Objective The aim of this study was to investigate the effects of Porphyromonas gingivalis(P.gingivalis,Pg)and Lactobacillus rhamnosus PROSCI-109(PROSCI-109,Pro)on RA.We analyzed the pathogenic mechanism of Porphyromonas gingivalis acting through the gut-joint axis to induce and exacerbate RA at multiple levels,including manifestation,pathological,inflammatory factors,anti-cyclic citrullinated peptide antibody,and macrogenome sequencing of intestinal bacteria;and explored whether the probiotic Lactobacillus rhamnosus could resist this pathogenic mechanism and alleviate arthritis by altering the intestinal flora,providing a basis for whether probiotics could be a new approach to treat RA.Methods(1)Twelve DBA-1 male rats were randomly divided into blank control group,collagen-induced arthritis(CIA)group,CIA rats given Porphyromonas gingivalis(CIA+Pg)group,and CIA rats given Porphyromonas gingivalis and probiotic Lactobacillus rhamnosus PROSCI-109 treatment(CIA+Pg+Pro)group.The CIA+Pg group and CIA+Pg +Pro groups were apply with P.gingivalis to induce periodontitis models,and the CIA+Pg+Pro group was given Lactobacillus rhamnosus PROSCI-109 intervention.After successful modeling,the joint morphology of each group was recorded every 4 days and the paw thickness was measured.Mice were killed at the end of the experiment,and pathological sections of the joint and small intestine tissues were stained to compare the pathological morphological changes.(2)The serum anti-cyclic citrullinated peptide antibody levels of each group were measured by ELISA to analyze whether P.gingivalis induced and aggravated RA through citrullinated modification,and to clarify whether Lactobacillus rhamnosus PROSCI-109 could inhibit citrullination modification.(3)The gene expression levels of pro-inflammatory factors IL-1β,IL-6,IL-17 A,TNF-α and anti-inflammatory factor IL-10 in mouse intestinal and joint tissues were detected by Quantitative Real-time PCR using primers designed from the NCBI database.The gene expression levels of TJP-1 and Ocln,which are related to intestinal permeability,were also detected in the intestinal tissues.(4)Fecal samples were collected from each experimental group of mice,and metagenomic sequencing was performed to analyze the changes of intestinal flora at three levels: gene,species and function.Results(1)Mice in the CIA+Pg group were the first to start experiencing apparent joint red and swollen and had higher degree of paw thickness than the other groups: CIA group vs CIA+Pg group,P(25 days)= 0.0294;P(45 days)= 0.0378.Arthritis scores were higher than the other groups: CIA+Pg group vs CIA+Pg+Pro group,P(25 days)= 0.0198;P(49 days)=0.0474.(2)The pathological results showed that the degree of synovial hyperplasia,the extent of synovial inflammatory cell infiltration,the number of osteoclast activation,the degree of bone and cartilage destruction,and the degree of intestinal mucosal edema in the CIA+Pg group were the most obvious changes among all groups;the above changes were reduced in the CIA+Pg+Pro group compared with the CIA+Pg group,and the differences between all groups were statistically significant.(3)In each group,the concentration of anti-cyclic citrullinated peptide antibody in the serum samples of mice was low.(4)P.gingivalis and Lactobacillus rhamnosus PROSCI-109 increased and decreased joints IL-1β and IL-6 gene expression levels,respectively.P.gingivalis increased intestinal IL-17 A expression and decreased IL-10 expression levels;Lactobacillus rhamnosus PROSCI-109 decreased intestinal IL-17 A expression and increased IL-10 expression level,and the difference was statistically significant.It was confirmed that P.gingivalis aggravated the tissue inflammatory response and Lactobacillus rhamnosus PROSCI-109 could reduce the inflammatory response.(5)P.gingivalis and Lactobacillus rhamnosus PROSCI-109 increased and decreased intestinal TJP-1 and Ocln gene expression levels,respectively,and the differences were statistically significant,indicating that these two bacteria affect intestinal permeability,with P.gingivalis playing a destructive role and Lactobacillus rhamnosus PROSCI-109 having a reparative role.(6)P.gingivalis and Lactobacillus rhamnosus PROSCI-109 regulate intestinal flora gene expression and affect intestinal flora composition;Lactobacillus rhamnosus PROSCI-109 can increase the proportion of Lactobacillus spp.of Lactobacillus taiwanensis and promote a variety of metabolic reactions.Conclusion(1)P.gingivalis is one of the environmental factors that induce aggravation of RA,advance the onset of arthritis,and aggravate RA by affecting the expression of inflammatory factors,increasing intestinal permeability,and affecting the composition and metabolism of intestinal flora.Lactobacillus rhamnosus PROSCI-109 can resist P.gingivalis,reconstitute the intestinal flora,regulate the expression of inflammatory factors,repair the intestinal mucosal barrier,and reduce the clinical manifestations and pathological damage in animal models.It attenuates clinical manifestations and pathological damage in animal models and may become a probiotic strain for the adjuvant treatment of RA. |