Mechanism Of α-mangostin Inhibiting Synovial Angiogenesis In Rheumatoid Arthritis Through Metabolic Correlation Rho/ROCK Pathway

Objective: Synovial angiogenesis is a driver of joint damage in Rheumatoid Arthritis(RA).We have found that α-Mangostin(α-MAN)which is rich in mangosteen,can effectively alleviate the experimental arthritis,which is mainly manifested as the reduction of joint injury and the recovery of metabolic-inflammatory feedback disorder.In view of the high metabolic environmental sensitivity of the Rho/ROCK pathway,it is important to promote angiogenesis under pathological conditions.In this study,we intended to determine that the joint protection of α-MAN is related to its anti-angiogenesis effect on the basis of further proving the therapeutic effect of α-MAN on RA.The regulation of energy metabolism induced by α-MAN stimulation is the direct factor for the improvement of local oxidative stress and hypoxic environment,and preliminarily elucidated the molecular mechanism of α-MAN in inhibiting the angiogenesis of synovial membrane in RA through the regulation of Rho/ROCK pathway.Methods: The research was carried out in two parts.The first part is the study on the role of Rho/ROCK pathway related to energy metabolism in RA accompanied by synovial angiogenesis.First,we selected human umbilical vein endothelial cells(HUVEC)for culture in vitro,and collected serum stimulating cells of RA patients as the model group,and set the non-stimulating control group.The cells of the two groups were cultured in a repeat group for 4 h and 24 h,respectively.Metabonomics was used to analyze the changes of intracellular metabolite levels and high-energy substances.Secondly,Resveratrol(RSV),an SIRT1 agonist,was added to stimulate cells and interfere with energy metabolism,and then biochemical kits and enzyme-linked immunosorbent assay(ELISA)were used to detect Glucose(Glu),Lactic Acid(LD)and transforming growth factor-β(TGF-β),interleukin-6(IL-6)and vascular endothelial growth factor(VEGF);Then the angiogenesis ability was evaluated by cell scratch test,tube-forming test and transwell test.Shikonin was added into the normal control group,LPS-stimulated model group and RSV-treated group for stimulation,and the cell supernatant was collected to detect the concentrations of Glu and PA.Further,Western Blot(WB)and RT-q PCR were used to detect the differences in protein and m RNA levels of metabolism,Rho/ROCK pathway and angiogenic factors,respectively.Bikinin was used to stimulate cells in an LPS-stimulated inflammatory environment to block energy utilization,and then biochemical kits,ELISA,WB and RT-q PCR were used to repeatedly detect the above indicators to evaluate the effects of regulatory metabolism on Rho/ROCK pathway and angiogenesis.Then,we interfered with the expression of hypoxia-inducible factor-1α(HIF-1α),Rho and ROCK in vitro,and judged the specific effect and mode of action of Rho/ROCK pathway on angiogenesis through ELISA,RT-q PCR and angiogenesis observation experiment.The second part is about α-MAN’s inhibition on synovial angiogenesis in RA patients via regulated energy metabolism and Rho/ROCK pathway.First,α-MAN was intragastrically administered to adjuvant arthritis(AIA)model rats,and then the plasma inflammatory factors TGF-β,IL-6,and angiogenesis factors HIF-1α and VEGF were detected by ELISA.The blood samples were subjected to metabolomics analysis,and then the joint sections of rats were subjected to immunohistochemistry.α-MAN was further treated in vitro with HUVEC cell model,and the oxidative stress indicators of Malonaldehyde(MDA)and Superoxide Dismutase(SOD)in the cell supernatant were detected using biochemical kits.Then,in the same way as in the first part,Bikinin and si-HIF-1α were respectively given to stimulate HUVEC cells to block energy utilization and HIF-1α pathways,and α-MAN stimulation was performed.Also,the relevant indicators were detected and the therapeutic effect of α-MAN was evaluated using the method in the first part.Results: Part I: First,through metabolomics monitoring at different times,we found that the concentrations of glucose-6-phosphate,PA and other glucose metabolic intermediates in the model group were significantly increased with the increase of culture time,while the concentrations of tricarboxylic acid cycle intermediates were not significantly changed.Besides,the concentrations of high-energy compounds(ATP,ADP,GTP and GDPand some key coenzyme related to energy metabolism in the model group were decreased after cell culture for 4 h.Biochemical kits and ELISA experiments showed that the content of Glu and PA in model group was decreased,while the production of LD was increased.However,RSV increased the concentration of Glu and PA but decreased that of LD.Later,in cell scratch test,tube forming test and transwell test,we found that the model group had strong angiogenesis ability,while the treatment group weakened or even approached the level of the normal group.After Shikonin stimulation,we found that the content of Glu and PA in Glu model group was decreased,and RSV addition also increased the concentration of Glu and PA.The WB and RT-q PCR assays showed that the protein and m RNA levels of 1(Silent Information Regulator 1(SIRT1)were significantly decreased in the model group and increased with the addition of RSV.p-Rho A,p-ROCK,and VEGF were significantly increased in the model group at protein and m RNA levels,but significantly decreased to nearly normal level with the addition of RSV.Bikinin was then used to stimulate and block energy utilization,and similar results were produced using the above tests.Next,simultaneous interference of HIF-1α and Rho/ROCK in the cellular inflammatory environment was detected by RT-q PCR and ELISA.It was found that although interference of HIF-1α had caused the increase of SIRT1 level,decreased expression of Rho A and ROCK,and significantly reduced VEGF level,but further interference of Rho/ROCK greatly deepened the effect.Part II: Compared with normal animals,inflammatory indicators such as TGF-β and IL-6,metabolic indicator LD,oxidative stress indicator MDA,and angiogenesis indicators HIF-1α and VEGF in AIA model rats were significantly increased,while α-MAN treatment significantly reduced the concentrations of TGF-β,IL-6,LD,MDA,HIF-1α and VEGF.Immunohistochemical experiments showed that the expressions of HIF-1α and VEGF in theα-MAN treatment group were less than those in the model group.In vitro cell experimentα-MAN therapy repeatedly tested inflammatory indicators such as TGF-β and IL6,metabolic indicator LD,oxidative stress indicator MDA,and angiogenesis indicators HIF-1αand VEGF and found that these indicators were all reduced after α-MAN treatment.PA content and Lactate Dehydrogenase(LDH)content leading to the conversion of PA to LD were decreased.Metabolomics testing of rat plasma showed increases glucose-6-phosphate,fructose-6-phosphate,glycerol-3-phosphate,and phosphoenolpyruvate in AIA rats,andα-MAN treatment significantly reduced concentration levels of all these metabolites;The in vivo and in vitro tests showed that α-MAN treatment significantly reduced the concentration of HIF-1α in the plasma of AIA model rats and in LPS-stimulated cells.Administration ofα-MAN in vitro revealed that it reduced the levels of Rho and ROCK in cells under inflammatory environment through WB and RT-q PCR tests.Besides,the strong angiogenesis ability in the model group was observed through scratch test,tube-forming test and transwell test,while this phenomenon was greatly reversed by α-MAN treatment.Conclusion: Part I: The metabolomics test results show that glucose metabolism,especially glycolysis,is in an accelerated state under inflammation,and energy consumption is increased.The kit test further proved that RSV could inhibit metabolism,especially the acceleration of glycolysis process.Besides,the changing trend of metabolites was unchanged after the addition of shikonin,which excluded the effect of NF-κB pathway on the glycolysis process.Inhibition of glycolysis by angiogenesis experiment could inhibit angiogenesis under inflammation.WB and RT-q PCR assays demonstrated that inhibition of metabolic processes inhibits Rho/ROCK pathway levels and angiogenesis under inflammation.Repeated experiments after Bikinin stimulation verified that the regulation of energy-manipulated metabolism could affect the level of Rho/ROCK pathway and angiogenesis under inflammation.The repeated experiments after silencing HIF-1αdemonstrated that Rho/ROCK pathway regulated angiogenesis independently of HIF-1αpathway.Part II: α-MAN not only has a good therapeutic effect on RA,but also protects the joints by inhibiting angiogenesis.α-MAN regulates the metabolic level by effectively inhibiting the core steps of oxidative phosphorylation such as glycolysis and tricarboxylic acid cycle,and reduces tissue oxygen demand,thereby relieving local joint hypoxia and blocking the activation of Rho/ROCK pathway induced by ROS,HIF-1α and other factors.α-MAN inhibits the angiogenesis process of RA synovial membrane regulated by Rho/ROCK pathway.