| Knee Osteoarthritis(KOA)is a degenerative disease commonly seen in middle-aged and elderly people.The main pathological manifestations are Knee cartilage degeneration,osteophyte formation at the joint,aseptic inflammation of surrounding soft tissues and secondary subchondral bone destruction.The symptoms of KOA are sexual pain,swelling,poor mobility,joint nooses and joint deformities of the knee joint.In severe cases,they can also cause disability.In China,the prevalence of KOA in adults is about 15%,and that in people over 60 years old is 50%,which is closely related to gender.In women,the prevalence of KOA gradually increases from 8.8%to 42.7%.Traditional Chinese medicine believes that the incidence of KOA is based on the loss of liver and kidney,Qi and blood deficiency,and the invasion of cold and dampness as the standard,which should be classified into the category of "bone Bi","jin Bi" and "pain Bi".Guizhi Shaoyao Decoction and Guizhi Shaoyao Zhimu Decoction have good effect on treating OA by isotherm meridian,activating blood circulation,and greatly reducing the side effects caused by oral western medicine.Cinnamaldehyde,the main effective component of Cinnamomum cassia,has anti-inflammatory,anti-bacterial,anti-oxidation,alleviating inflammation of synovial membrane and chondrocyte,and protecting cartilage.At present,in addition to surgery and minimally invasive treatment,patients need long-term oral non-steroidal anti-inflammatory drugs for pain relief.Long-term medication has caused varying degrees of side effects on the gastrointestinal,liver and kidney functions of patients,which impair the overall function of patients.Therefore,many patients cannot continue to take medication and can only tolerate pain.The study found that patients with KOA had gut microbiota dysregulation,as well as dysregulation of amino acid metabolism in blood and urine.Therefore,it is of great significance to further study the status of intestinal flora in patients with KOA and maintain the diversity of intestinal flora in patients with KOA while alleviating joint inflammation,reducing the side effects of treatment and delaying the disease progression.Objective:1.To study the gut microbiota and metabolism of patients with knee arthritis and then find out the different microorganisms and metabolites.2.To study the effect of cinnamaldehyde on knee arthritis and its regulation on intestinal flora.3.The effect of cinnamaldehyde on macrophage polarization and its correlation with intestinal flora were preliminarily investigated.Methods:1.The differences in gut microbiota and metabolites between patients with KOA and normal subjects were observed in clinical trials.Fecal samples from patients with KOA and normal subjects were selected respectively.The gut microbiota and metabolic diversity of the two groups were detected by 16S rRNA high-throughput sequencing technology and non-target metabolomics technology,and the different microorganisms and related metabolites in the two groups were found out.2.Animal experiments were conducted to investigate the immune mechanism of cinnamaldehyde regulating gut microbiota and intervening in the treatment of KOA.Koa rabbit model were established by intraarticular injection of papain protein into knee joint,and randomly divided into four groups:blank group,experimental group,control group and model group.The experimental group was intragastrically gavaged with cinnamaldehyde,while the control group was intragastrically gavaged with diclofenac sodium sustained-release capsule.The model group was not given any natural feeding intervention after modeling,while the blank group was given no modeling and no intervention.Samples were collected after 4 weeks of treatment.①The fecal samples of rabbits in each group were collected and the abundance and diversity of gut microbiota were detected by 16S rRNA high-throughput sequencing technology.②The pathological manifestations of synovial tissue in each group were observed under light microscope.③The contents of inflammatory factors including TNF-α,IL-12 and IL-10 in rabbit joint fluid were determined by Elisa.④The expressions of iNOS and Arg-1 in M1 and M2 macrophages in synovial tissues were detected by immunohistochemistry and Western Blot.⑤The levels of M1 and M2 macrophage markers CD 11c and CD206 in rabbit articular fluid were detected by reverse transcription polymerase chain(RT-PCR).Results:1.To observe the difference of intestinal flora and metabolism between KOA patients and normal people by using Clinical trial.1.1 Gut microbiota test results:There were significant differences in patients among Ruminococcaceae,Gemmiger,Actinomyces and Actinomyces in normal subjects compared with patients;these microorganisms may be related to joint injury.1.2 After screening,the three dipeptide compounds were significantly different between KOA patients and normal subjects:Iso-Pro,Phe-Val,Phe--Iso.2.Study the immune mechanism of cinnamaldehyde on koa by regulating intestinal flora in vivo.2.1.Elisa showed that both cinnamaldehyde and diclofenac sodium could reduce the levels of TNF-α and IL-12 in rabbit knee joint fluid,but the effect between the two groups shows no significant difference;The level of IL-10 was increased,but the effect between the two groups shows no significant difference.2.2.Immunohistochemistry showed that cinnamaldehyde could decrease the mean optical density of iNOS,a marker of M1 macrophages,increase the optical density of Arg-1,a marker of M2 macrophages,and promote the polarization of M1 macrophages to M2.It also had a stronger effect on regulating M1/M2 macrophage polarization than the control group,The difference between them was statistically significant.2.3 Western Blot experiments demonstrated that cinnamaldehyde could reduce the expression of iNOS,a marker of M1 macrophages,increase the expression of Arg-1,a marker of M2 macrophages,and promote the polarization of M1 macrophages to M2.The cinnamaldehyde had a stronger effect on regulating M1/M2 macrophage polarization than the control group.The difference between them was statistically significant.2.4.RT-PCR experiments demonstrated that cinnamaldehyde could down-regulate the level of CD11c gene,a marker of M1 macrophages,up-regulate the level of CD206 gene,a marker of M2 macrophages,and promote the polarization of M1 macrophages to M2.There was no significant difference in the expression of CDllc and CD206 genes by diclofenac sodium in the control group,and the polarization effect of macrophages was not significant.2.5 The results of gut microbiota①In the diversity comparison of intestinal flora,there were differences among groups:normal group>experimental group>control group>model group.②Compared with the blank group,the abundances of Chloroplast,Streptophyta,Butyricimonas,Comamonadaceae and Odoribacteraceae in the model group were increased,indicating that the incidence of arthritis was related to the increase of the abundance of the above species;③Compared with the model group,the abundance of Veillonellaceae,Phascolarctobacterium and Peptococcaceae in the experimental group was increased,suggesting that cinnamaldehyde can relieve joint inflammation by stabilizing the abundance of the above bacteria;④Compared with the model group,the experimental group and the blank group showed an increase in the abundance of Veillonellaceae and Phascolarctobacterium,indicating that cinnamaldehyde could restore the intestinal status of the model group to an approximate normal level,and Veillonellaceae and Koala could be considered as the hallmark microorganisms for the improvement of the model group flora from dysregulation to improvement.⑤Bacteroidetes and Firmicutes were correlated with macrophage markers and inflammatory factors,indicating that both of them were also involved in macrophage polarization.Conclusion1.Gut microbiota and their metabolites in patients with KOA are significantly different from those in normal subjects,and the increased abundance of chloroplast,Trichoderma,Comamonas,Odoraceae,and Butyricomonas may be related to inflammation of the knee joint.2.The pathogenesis of KOA,which is the result of a combination of glucose metabolism,lipid metabolism and immune regulation,may be associated with excessive production of short-chain fatty acids.3.Cinnamaldehyde can modulate M1/M2 macrophage polarization and alleviate inflammation of synovial tissue in the joint by stabilizing Weronellaceae,Koalensis,and Peptococcaceae in rabbits. |
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