| Objective: Metabolic disorders are common in patients with rheumatoid arthritis(RA).Qing Luo Yin(QLY)is a representative prescription for the treatment of heat arthromyodynia in traditional Chinese medicine.Our team’s previous studies have shown that QLY can effectively reduce the development of inflammation in various arthritis models,and has anti-inflammatory effects both in vivo and in vitro.Meanwhile,it can effectively restore the metabolic disorders in the animal arthritis models.In this study,we investigated the involvement of fatty acids in RA-related inflammation,and attempted to prove that QLY can restore the immune homeostasis by affecting fatty acids levels.Methods: Using different arthritis models,we tested the effects of HFD and fatty acids supplement on RA.Experimental animals were weighed and the arthritis score was evaluated periodically.Disease severity was evaluated by foot morphology and HE sections,inflammatory factors,oxidative stress product levels,monocyte phenotype,metabolomics and metabolism-related indicators.In vitro cell experiments,CO-IP and dual-luciferase reporter gene experiments were used to validate the impacts of fatty acids on PPARγ and its downstream signals.On the basis of the above mechanism study,AA model was established and 10g/kg QLY(calculated by raw drug dose)was used to treat arthritis,in order to prove that QLY may interfere with FA metabolism to treat arthritis.The observation lasted for 30 days,and the rats were killed on the 31 st day.The characteristics of QLY therapy for AA and the involved targets and pathways were preliminarily determined by observing the morphology of foot paws and HE tissue sections,and measuring the relevant inflammatory and biochemical indexes.The results were verified by QPCR,WB and other methods.Relevant conclusions were verified by cell models in vitro.Results: 1.Under the condition of energy restriction,the arthritis score of rats in the HFD group was significantly reduced,the joint damage was improved,and the serum levels of TNF-α and IL-1β in the HFD group were also decreased.Metabolomics monitoring at different stages showed that lactate was the key differential metabolite between the two groups.The serum lactate level in the HFD group decreased significantly on day 28 and day35.2.Without energy restriction,the joint diameter of HFD-fed AIA mice gradually decreased after modeling,which was significantly lower than that of the AIA group.The knee joint injury was alleviated,and the levels of IL-1β and Monocyte Chemoattractant Protein-1(MCP-1)in serum were also decreased.The increased Glucose(GLU)level and the proportion of non-classical monocytes significantly increased the expression of PPARγ in adipose tissue.3.GC-MS results showed that on days 14 and 21,the levels of Palmitic Acid(PA),Stearic Acid(SA),Oleic Acid(OA)and Linoleic Acid(LA)were significantly decreased in plasma of AA rats.HFD feeding can increase the concentration of these four kinds of FA,and the changes of Unsaturated Fatty Acid(USF)are more obvious.4.Supplementation of four HFD-associated FA can alleviate the condition of AA rats: Saturated Fatty Acid(SF),USF,Mixed Fatty Acid(MF).The levels of IL-1β and PGE2 in the serum of rats were decreased,and the levels of IL-6 were significantly decreased by USF and MF supplementation.5.In vitro experiments demonstrated that USF stimulated PPARγ pathway more than SF,SF stimulated TLR4 pathway,but USF could effectively avoid the direct activation of TLR4.6.QLY can reduce the levels of MCP-1,IL-1β and IL-6 in serum.It is preliminarily speculated that QLY has a good therapeutic effect on the inflammatory response controlled by monocytes/macrophages,and QLY treatment can promote the Silent Information Regulator 1(SIRT1)and PPARγ, suggesting a potential effect of QLY on FA metabolism.7.QLY treatment can restore the perirenal fat reserve to a certain extent,increase the volume of adipocytes,reduce inflammatory infiltration,reverse the decline of serum adipokines in AA rats,restore the changes in oxidative stress indicators,and inhibit the differentiation of pro-inflammatory macrophages in White Adipose Tissue(WAT).8.In vitro experiments show QLY can improve the fat cells before SCD-1,PPARγ,SIRT1 protein expression,and Nicotinamide phosphoribosyltransferases transferase(NAMPT)expression is decreased obviously.The expression of PPARγ decreased after LPS stimulation in preadipocytes,but increased significantly after the stimulation of serum containing QLY,which was subsequently reversed by PPARγ selective inhibitor T0070907.9.In vitro experiments,inhibition of FA intake in THP-1 cells by blocking the CD36 pathway significantly reduced the intracellular FA and PPARγ expression,while QLY stimulation increased FA concentration and PPARγ expression.The intracellular FA content was similar by adding excessive FA,and there was no significant difference in PPARγ expression among the groups.After silencing PPARγ pathway in THP-1 cells,it was observed that the levels of TNF-α,IL-1β and IL-6 were significantly increased after the serum stimulation of AA rats.The addition of QLY could reduce the levels of TNF-α,IL-1β and IL-6,and the levels increased again after silencing PPARγ pathway.Conclusion: 1.Regardless of the difference in total intake,HFD inhibited inflammatory progression in different arthritis models.2.At the peak of inflammation in AA model rats,the contents of four FA related to HFD were significantly decreased,and the reduction of USF was more obvious.3.The four FA supplementation derived from HFD can alleviate the condition of AA rats,and USF can alleviate the condition of AA more significantly than SF supplementation.This therapeutic activity is related to PPARγ and NF-κB signaling pathways.4.QLY treatment can effectively inhibit the inflammatory progression in the acute inflammatory stage of AA rats and restore the up-regulation of FA concentration.5.QLY activates PPARγ signaling pathway by regulating FA concentration,improves immune and metabolic microenvironment in vivo,and reverses the pathological functional changes of AA inflammation. |
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