Role Of Macrophage Derived ?-arrestin-1 And Cannabinoid Receptor 2 In Ulcerative Colitis

The number of ulcerative colitis patients tends to be increased year by year throughout the world.In the United States,the cost spent in the treatment of ulcerative colitis is about 81-149 billion dollars every year,while in Europe,the number is up to 29.1billion euro.With the improvement of the living standard,the incidence of ulcerative colitis in our country is increasing.Since the pathogenesis of ulcerative colitis is not exact,all drugs available can only relieve the symptoms but cannot completely cure it,which bring a heavy economic burden and mental pressure to society and the families of patients.What's more,the lasting colitis is likely to finally develop into colitis-associated cancer,so clarifying the pathophysiological mechanism of the occurrence and development of ulcerative colitis,and exploring new drugs to prevent and treat ulcerative colitis is extremely urgent.Here,this paper is divided into the following two parts.In the first part,we explored the role of macrophage ?-arrestin-1 in ulcerative colitis and the mechanism underling ?-arrestin-1 expression.It has been reported that global knockout of?-arrestin-1 protected TNBS and DSS-induced colitis while non-hematopoietic ?-arrestin-1knockout aggravate the severity of DSS-induced colitis which means ?-arrestin-1 from different cells exhibits distinct role on colitis.We have cultured myeloid ?-arrestin-1conditional knockout mice(?-arrestin-1floxp/floxpLysM-Cre)using Cre-LoxP recombinase system and found that ?-arrestin-1floxp/floxpLysM-Cre mice behaved more resistance to DSS-induced colitis with reduced body weight loss and bloody stool score,improved colon inflammation,indicating that myeloid ?-arrestin-1 could promote colitis.In peritoneal macrophage from wild type and ?-arrestin-1floxp/floxpLysM-Cre mice,the level of inflammatory cytokines induced by TNF-? was decreased in ?-arrestin-1floxp/floxpLysM-Cre group compared to wild type group.To explore the mechanism of ?-arrestin-1 regulating inflammatory cytokines production,we used western blot,immunofluorescence and EMSA and discovered?-arrestin-1 could augment the activation of NF-?B and MAPK pathway induced by TNF-?.Furthermore immunoprecipitation revealed that ?-arrestin-1 direct combined with IKK and I?B?.In addition,in the murine model of colitis induced by DSS,we found that ?-arrestin-1was increased significantly.In cultured macrophages,TNF-? stimulation also increased the expression of ?-arrestin-1.Dual luciferase assay showed that NF-?B may mediate ?-arrestin-1gene transcription.To verify this hypothesis,we used BAY11-7082,a NF-?B activation inhibitor,to inhibit the activation of NF-?B and found the mRNA and protein level of?-arrestin-1 induced by TNF-? were suppressed notably,which confirmed that NF-?B couldregulate the expression of ?-arrestin-1 in macrophage.Taken together,we demonstrate that?-arrestin-1 promotes the production of inflammatory cytokines through NF-?B and MAPK signaling pathway in macrophage which results in the aggravating of colitis.Meanwhile,the elevated level of ?-arrestin-1 in macrophage is mediated by NF-?B.Our results may provide a new strategy for the treatment of ulcerative colitis.In the second part we focus on the role of cannabinoids receptor 2(CB2)and autophagy on ulcerative colitis.Activation CB2 receptor can produce anti-inflammatory effects,which illustrated by an article from our laboratory revealing that activation of CB2 receptor reduced inflammatory cytokine production,inhibited synovial cell proliferation,reduced joint bone destruction and leukocytes infiltration,thereby protecting mice from rheumatoid arthritis.Autophagy is an important self-protective mechanism and can provide energy for cell with challenges which participate in proliferation,differentiation and death of cells.Autophagy can effectively attenuate the over-triggering of several self-defensive pathways such as inflammatory reaction and immune responses thus plays an important role in a variety of diseases such as cancer,infection,neurodegenerative and inflammatory diseases.In our previous work,we demonstrated that activation of CB2 receptor could promote autophagy and inhibit NLRP3 inflammasome in microglia leading to the alleviation of experimental autoimmune encephalomyelitis.In this work,we found that treatment of HU308,a selective CB2 receptor agonist,attenuated the symptoms of DSS-induced colitis in mice while CB2 receptor knockout mice were more susceptible to colitis.Activation CB2 receptor inhibited the expression of NLRP3,Casp-1 p20/Casp-1p45 ratio,proIL-1? and increased autophagy related protein Beclin-1 and LC3-II/LC3-I level,reduced SQSTM1 in colon tissues of DSS mice and peritoneal macrophage induced by LPS/DSS.Compared to wild type group,CB2 receptor knockout led to more robust expression of NLRP3,Casp-1 p20/Casp-1 p45 ratio,proIL-1? and decreased level of autophagy both in vivo and in vitro.In order to verify the role of autophagy in the inhibitory effect of CB2 receptor on NLRP3 inflammasome,we used autophagy targeted gene 5(Atg5)small interfering RNA(siRNA)to block autophagy in RAW264.7 and found Atg5 si RNA partly abolished the inhibition of HU 308 on NLRP3 inflammasome.In addition,the autophagy inhibitor 3-methyladenine(3-MA)also weakened the protective effect of HU 308 on DSS-induced colitis and the inhibitory effect on NLRP3 inflammasome.Finally,we detected the autophagy related signaling pathway,and proved that AMPK-mTOR-P70S6 K mediated the inhibitory effect of CB2 receptor on NLRP3 inflammasome.These results together showed that activation of CB2 receptor can inhibit NLRP3 inflammasome by increasing autophagy,which play a protective role in the colitis.In conclusion,CB2 receptor and autophagy is expected to become a new strategy and target in the treatment of ulcerative colitis.