| BackgroundRheumatoid arthritis(RA)is an autoimmune disease with synovitis.With the extension of the course of the disease,the synovium invades and degrades the cartilage matrix promoting joint destruction.Massive hyperplasia of FLS(Fibroblast-like Synoviocytes)is one of the hallmarks of RA.FLS not only strongly respond to a pro-inflammatory environment,but also actively and autonomously drive joint inflammation.Besides,the immune cells and pro-inflammatory factors also play an essential role in regulating the microenvironment in the joint cavity and can not be ignored in the treatment of RA.Some studies have found that the level of glucose metabolism is elevated in RA-FLS,and a variety of glycolytic-related enzymes also express highly,which similar to the Warburg effect in a tumor.Meanwhile,with the increase of glycolytic metabolites,such as lactic acid or pyruvate,the synovial tissue is chronically in a hypoxic environment,which stimulates the expression of inflammatory factors like IL-6 or TNF-?,and promotes cell proliferation.Our team has used the technology of glucose metabolism matrix chip(Glucose Metabolism RT2 Profiler TM PCR Array)to screen genes related to glucose metabolism,and the results showed that HK2(Hexokinase 2),ENO1(Enolase 1)and PGK1(Phosphoglycerate kinase 1)was high specificity in the synovial tissue collagen-induced arthritis rats.Accordingly,glycolysis plays an essential role in the development of RA.2-Deoxyglucose(2-DG),a glucose analog,has the 2-hydroxyl group replaced by hydrogen and can be transferred by glucose transporters(GLUTs).2-DG competes with glucose for HK2.And the phosphorylated product of 2-DG is 2DG-6-P(2-Deoxy-D-glucose-6-phosphate),which cannot be further metabolized through glycolysis.At present,2-DG has been widely used in tumor research and has been confirmed that it can effectively reduce ATP synthesis,which further inhibits mTOR(mammalian target of rapamycin)signaling pathway in tumor cells.As a regulatory hub for protein synthesis in the body,the mTOR signaling pathway is abnormally active in a variety of cancers,auto-inflammatory,and autoimmune diseases.Studies have found that mTOR also markedly increases in RA-FLS cells and participates in cell invasion.Given the similar pathological environment between tumor cells and RA-FLS,we think that 2-DG may exert its inhibitory effect on RA-FLS cell growth by targeting the mTOR signaling pathway through inhibiting glycolysis.Our study was conducted in two parts.The first part was cell experiments.Human synovial tissues were acquired from the knee joints of patients with RA during surgery and then were cultured for 3-5 passages for further study.2-DG was dissolved in Phosphate-buffered Saline and then diluted into different concentrations.After RA FLSs were treated with 2-DG,we could observe differences between 2-DG treatment groups and the control group in cell proliferation,apoptosis,migration,and cytokine secretion.The second part was animal experiments.By injecting bovine ? collagen into rats,we built the classical model of rheumatoid arthritis.The treatment group was given 5 mg/kg 2-DG using intraperitoneal injection.The arthritis clinical scoring curve was used to evaluate the degree of inflammation in the model.By detecting the expression of key glycolytic enzymes and the mTOR signaling pathway,the change of immune cells,and the secretion level of inflammatory factors,we could explore the therapeutic mechanism of 2-DG in CIA rats.Methods1.Clinical specimens of synovial tissue were collected from RA patients(n=10)for culturing primary cells.2.RA-FLS cells were divided into the control group and treatment groups with 2-DG(1,10,20 mM).The cell proliferation ability of each group was detected using a CCK-8 kit.3.RA-FLS cells were divided into the control group and treatment groups with 2-DG(1,10,20 mM)for 48 h.Flow cytometry was used to detect cell apoptosis and inflammatory cytokines.Cell wound healing assay and a Transwell test were used to detect cell migration.4.RA-FLS cells were divided into the control group and treatment groups with 2-DG(10,20 mM)for 48 h.Real-time PCR was used to detect the mRNA levels of glycolysis-related enzyme genes,including HK2,PFK,TPI1,PGK1,ENO1,PKM,and LDH.The ATP kit was used to detect changes in ATP among these groups.Western blot was used to analyze the expression levels of HK2 and mTOR signaling pathway-related proteins,which helps to observe the effects of 2-DG on glycolysis and the mTOR pathway in RA-FLS cells.5.The CIA(Collagen-induced arthritis)rat model was established,and 2-DG was intraperitoneally injected into the CIA rat.The normal control group was composed of healthy rats.The curve of the clinical arthritis score was plotted over time.Also,histochemical pictures and a joint radiograph was used to observe the pathological changes of joint tissues.6.Real-time PCR was used to detect the mRN A levels of glycolytic enzyme genes HK2,PFK,TPI1,PGK1,ENO1,PKM,and LDH in the synovial tissues of NC group,CIA group,and 2-DG treatment group.Western blot was used to analyze the expression levels of HK2 and mTOR signaling pathway-related proteins in the NC group,CIA group,and 2-DG treatment group.Furthermore,the ATP kit was used to detect changes in ATP among these three groups(NC group,CIA group,and 2-DG treatment group).These tests help to observe the effects of 2-DG on glycolysis and the mTOR pathway in CIA rats.7.The proportion of B cells,CD4+T cells,CD8+T cells,NK(natural killer cell)cells,and Treg(regulatory cell)cells in the peripheral blood of these three groups(NC group,CIA group,and 2-DG treatment group)were detected by flow cytometry technology,to observe the effect of 2-DG on immune cells in CIA rats.8.The serum levels of IL-2,IL-4,IL-6,IL-10,IFN-y,and TNF-? in peripheral blood of rats were detected by flow cytometry in NC group,CIA group,and 2-DG treatment group,to observe the effect of 2-DG on cytokines in CIA rats.Results1.CCK-8 showed that the cell proliferation ability of RA-FLS treated with 2-DG was significantly reduced(P<0.001).These results illustrated that 2-DG could inhibit the proliferation of RA-FLS.2.Compared with the blank control group,the apoptosis rate of RA-FLS treated with 2-DG was increased remarkably(P1 mM=0.02,P10 mM<0.001,P20 mM<0.001),which showed that 2-DG could promote the apoptosis of RA-FLS.3.The function of cell migration was verified by the cell scratch test and the Transwell test.The cell scratch test represented that the healing ability of RA-FLS in the 2-DG treatment groups were suppressed heavily(P1 mM<0.01,P10 mM<0.001,P20 mM<0.001).Furthermore,the transwell experiment displayed that there was no change in the 2-DG treatment group with the lowest concentration(P1 mM=0.07).However,the cell migration ability of RA-FLS in the 2-DG treatment groups with the medium or the highest concentration was inhibited significantly(P10 mM<0.01,P20 mM<0.01).These results illustrated that 2-DG inhibits the migration of RA-FLS cells.4.Flow cytometry was used to detect the level of cytokines between the 2-DG treated group and the control group.Compared to the control group,there were no changes in the level of IL-2,IL-4,IL-10,and IFN-?(PIL-2=0.71,PIL-4=0.22,PIL-10=0.17,PIFN-?-0.78)in the 2-DG treatment groups.Only the level of IL-6(P1 mM<0.05,P10mM<0.01,P20 mM<0.01)and TNF-?(P1 mM=0.25,P10 mM<0.01,P20 mM<0.01)considerably dropped.These results indicated that 2-DG inhibited the secretion of IL-6 and TNF-?in the supernatants of RA-FLS.5.Real-time PCR was used to detect the effect of 2-DG(10,20 mM)on the mRNA levels of HK2,PFK,TPI1,PGK1,ENO1,PKM,and LDH in RA-FLS cells.The results showed that there was no difference of TPI1,PGK1,ENO1,and LDH among groups(PTPI1=0.676,PPGK1=0.679,PENO1=0.788,PLDH=0.272).Compared with the control group,the mRNA levels of HK2(P10 mM=0.042,P20 mM=0.011),PFK(P10 mM<0.001,P20 mM<0.001),PKM(P10mM<0.001,P20 mM<0.001)were significantly reduced in 2-DG treatment.The protein expression level of HK2 in the 2-DG treatment groups was limited(P10mM<0.01,P20 mM<0.01),and the intracellular production of ATP in the 2-DG treatment groups was also reduced(P10mM=0.019,P20 mM<0.001).These results illustrated that 2-DG could inhibit glucose metabolism in RA-FLS.6.The activation level of p-4EBP1 in the mTOR signaling pathway was notably up-regulated(P10mM<0.01,P20 mM<0.01).However,the expression of mTOR(Pio mM<0.01,P20 mM<0.01)and p-mTOR(P10mM<0.001,P20 mM<0.01)were significantly suppressed in the 2-DG treatment groups.These results illustrated that 2-DG could inhibit the mTOR pathway in RA-FLS.7.The clinical arthritis score demonstrated that the degree of joint damage in the CIA group was much higher than that in the NC group(P<0.001).The joint symptoms in the 2-DG group were significantly relieved compared with the CIA group(P=0.004).Joint swelling was more pronounced in the CIA group than that in the 2-DG group.The HE staining of joint indicated that bone destruction,synovial cell proliferation,and immune cell infiltration were evident in the CIA group.The radiograph of the CIA group displayed the disordered joint structure,bone erosion,and bone destruction.In contrast,there were not the above pathological changes in the 2-DG group.All of these results illustrated that 2-DG has a therapeutic effect on the joints of CIA rats.8.Real-time PCR detected the transcription levels of HK2,PFK,TPI1,PGK1,ENO1,PKM,and LDH in synovial tissue.The results showed that there were no differences of TPI1 and PGK1 among these three groups(PTPI1=0.187,PpGK1=0.110).The mRNA levels of HK2,PFK,ENO1,PKM,and LDH of the CIA group were significantly higher than those of the NC group(P<0.001,P<0.001,P<0.001,P<0.001,P<0.001).The mRNA levels of HK2,PFK,ENO1,PKM,and LDH in the 2-DG treatment group were significantly lower than those in the CIA group(P<0.001,P=0.029,P<0.001,P<0.001,P=0.017).In the 2-DG treatment group,only the mRNA level of LDH was much higher than that of the NC group(P<0.001).The mRNA levels of HK2,PFK,ENO1,and PKM were not different from the NC group(P=0.930,P=0.105,P=0.054,P=0.717).The expression of HK2 in the CIA group was markedly higher than that in the NC group(P=0.031).Instead,the expression of HK2 in the 2-DG treatment group was significantly lower than that in the CIA group(P=0.004).However,there was no difference between the 2-DG group and the NC group(P=0.197).Interestingly,the output levels of ATP had a similar trend as HK2 among these three groups.The expression of ATP in the CIA group dramatically increased(P<0.001).The expression of ATP in the 2-DG treatment group significantly declined(P<0.001),but there was no significant change compared with the NC group P=0.05 8).These results showed that 2-DG could inhibit the glucose metabolism in CIA rats.9.The results of each protein in the mTOR pathway were as follows.For the expression of mTOR,there was no significant difference between the CIA group and the NC group(P=0.354).The expression of p-mTOR in the CIA group was outstandingly higher than that in the NC group(P=0.016),but the p-4EBP1 was significantly lower than the NC group(P<0.001).Compared with the CIA group(P=0.568)and the NC group(P=0.076),the expression of mTOR in the 2-DG treatment group was not changed.The p-mTOR in the 2-DG group was significantly lower than that in the CIA group(P<0.001),but there was no difference compared with the NC group(P=0.098).The p-4EBP1 in the 2-DG group was significantly higher than the CIA group(P=0.027)but still lower than the NC group(P=0.021).These results showed that 2-DG could play a protective role in the joints of CIA rats by regulating the mTOR signaling pathway.10.With the treatment of 2-DG,the B cells in the blood of the CIA group significantly increased compared to the NC group(P=0.008).The percentage of the B cells in the 2-DG group was noticeably lower than that in the CIA group(P<0.001),but there was no difference compared to the NC group(P=0.074).The proportion of CD4+T cells in the CIA group was remarkably higher than that in the NC group(P=0.009).The ratio of CD4+T cells in the 2-DG group was markedly declined compared with the CIA group(P<0.001).There was no difference in the ratio of CD4+T cells between the 2-DG group and the NC group(P=0.109).The proportion of CD8+T cells in the CIA group is similar to the NC group(P=0.092).The ratio of CD8+T cells in the 2-DG treatment group significantly dropped compared with the CIA group(P=0.014)and the NC group(P<0.001).The differences in the proportion of NK cells between the CIA group,and the NC group were not obvious(P=0.091).The percentage of the NK cells in the 2-DG treatment group was significantly lower than that in the CIA group(P<0.001)and the NC group(P<0.001).The proportion of Treg cells in the CIA group was significantly lower than that in the NC group(P<0.001).The percentage of Treg cells in the 2-DG treatment group was significantly higher than that in the CIA group(P<0.001),but there was no difference compared with the NC group(P=0.063).All these results illustrated that not only could 2-DG inhibit the levels of B,CD4+T,CD8+T,and NK cells of CIA rats,but also promote the level of Treg cell in peripheral blood.11.Flow cytometry showed that there was no difference in the level of IL-2,IL-4,IL-10,and IFN-? among the three groups of NC,CIA,and 2-DG treatment groups(PIL-2=0.188,PIL-4=0.077,PIL-10=0.11,PIFN-?=0.067).The serum levels of IL-6(P<0.001)and TNF-?(P<0.001)in the CIA group were significantly higher than those in the NC group.The serum levels of IL-6(P=0.011)and TNF-?(P=0.002)in the 2-DG treatment group were significantly lower than those in the CIA group.There was no difference in the levels of IL-6(P=0.751)and TNF-?(P=0.689)between the 2-DG treatment group and the NC group.These results showed that 2-DG treatment could inhibit the secretion of IL-6 and TNF-?.Conclusions2-DG can relieve RA by inhibiting glycolysis in RA-FLS.Through inhibiting the expression levels of HK2,PFK,and PKM,2-DG can inhibit glycolysis,which decreases the production of ATP.2-DG can promote the apoptosis of RA-FLS,inhibit cell proliferation and migration,and the secretion of IL-6 and TNF-? via inhibiting the mTOR signaling pathway in RA-FLS and the synovium of CIA rats.2-DG can also increase the level of Treg cells in CIA rats,reduce the levels of B,T,and NK cells.The above results suggest that HK2 may be a potential target for RA treatment,and 2-DG may be a potential strategy for RA therapy by inhibiting glycolysis. |
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