| The CO released from CORM-2 has been proved to be anti-inflammatory, but the exact mechanism thereof is skill unknown. The previous study has shown that CORM-2 can activate Nrf2, which is closely related to organism anti-inflammation. Therefore, we assumed that the anti-inflammatory function of CORM-2 comes from its activation of Nrf2 pathway.In the vitro experiment, we compared CORM-2’s inhibiting effect on pro-inflammatory cytokines(TNF-α, IL-1β and IL-6) and iNOS in primary peritoneal macrophages derived from the wild type and Nrf2 knockout mice. In the vivo experiment, given non-lethal dose of LPS to the wild type and Nrf2 knockout mice, we examined the difference of CORM-2’s inhibiting effect on pro-inflammatory cytokines, iNOS and ICAM-1 in both the liver and brain tissues of the wild type and Nrf2 knockout mice. Also did we examined the difference of the inflammatory cells infiltration in both the liver and brain tissues of the wild type and Nrf2 knockout mice in HE staining. Besides, given lethal dose of LPS to the wild type and Nrf2 knockout mice, we studied CORM’s influence on their survival time.The result shows that CORM-2 has dramatically inhibited the expression of pro-inflammatory cytokines in the wild type mice, but not in the Nrf2 knockout mice in vivo and in vitro; also CORM-2 has substantially extended the survival time of the wild type mice, but not of the Nrf2 knockout mice. Thus, it can be concluded that Nrf2 is indispensable for CORM-2’s anti-inflammatory effect.Part 1 Carbon monoxide(CO) liberated by CO-releasing molecule CORM-2 activated Nrf2 pathway leads to protection against LPS-induced inflammation (In vitro)Objective:Although the anti-inflammatory effect of carbon monoxide has been proven by increasing researches, the detailed mechanism is not clear currently. In this study, an inflammatory response model was established by using LPS to stimulate primary peritoneal macrophage, and the relationship between the CORM-2’s anti-inflammation and the Nrf2 pathway was studied.Method:Establishing the optimum concentration of CORM-2 exrets anti-inflammation effects and the activation of the Nrf2 pathway at first, and then dedermining the relationship between the CORM-2’s anti-inflammation effects and the Nrf2 pathway. Isolating peritoneal macrophages from the wild type and the Nrf2 knockout mice, and the wild type mice divided into four groups:the control group, the CORM-2 group, the LPS group, and the LPS+CORM-2 group; and the Nrf2 knockout mice as before. The expression of pro-inflammatory cytokines such as TNF-a, IL-1β, IL-6 and iNOS from the wild type and Nrf2 knockout mice detectd by RT-PCR.Results:The optimal concentration of CORM-2 was 50μM. CORM-2 induced the Nrf2 nuclear translocation and the expression of the Nrf2 downstream genes including HO-1, NQO-1 and y-GCSm. The expression of pro-inflammatory cytokines ins control group and CORM-2 group of the wild type and the Nrf2 knockout mice were low. For the wild type mice, compared with the control group, the pro-inflammatory cytokines including TNF-a,IL-1β,IL-6 and iNOS in LPS group were substantially improved; and compared with LPS group, expression of the pro-inflammatory cytokines in LPS+CORM-2 group were substantially reduced (P<0.05). And for the Nrf2 knockout mice, compared with control group, the expression of the TNF-α,IL-1β, EL-6 and iNOS in the LPS group were obviously enhanced; and the expression of the pro-inflammatory cytokines in the LPS+CORM-2 group was not reduced obviously.Conclusions:The mechanism of carbon monoxide’s anti-inflammation has not been elucidated; however, the Nrf2-ARE pathway is closely related to the oxidative stress and inflammation, so that the relation between the anti-inflammatory effect of the carbon monoxide and the Nrf2-ARE pathway is worthy of our exploration. We have shown that CORM-2 can suppress the expression of pro-inflammatory cytokines, like TNF-a, IL-1β, IL-6 and iNOS of the wild type mice, but not the Nrf2 knockout mice. Therefore, it can be speculated that Nrf2 is essential for carbon monoxide’s anti-inflammation.Part 2 Carbon monoxide(CO) liberated by CO-releasing molecule CORM-2 activated Nrf2 pathway leads to protection against LPS-induced inflammation (In vivo)Objective:We have proven that the Nrf2 pathway is essential for CORM-2’s anti-inflammation in vitro. However, it is still unknown whether the situation in vivo is similar to the results of the experiment in vitro. In this study, the wild type and Nrf2 knockout mice were administrated a sublethal dose of LPS so as to establish a sepsis model, and the expression of pro-inflammatory cytokines of the wild type and Nrf2 knockout mice after CORM-2 treatment were compared.Methods:Sepsis model was established by intraperitoneal injection of LPS.24 wild type and 24 Nrf2 knockout mice were provided respectively. The wild type mice were randomly divided into four groups:the control group; CORM-2 group (CORM-230mg/kg); LPS group (LPS,10mg/kg); CORM-2 intervention group(LPS 10mg/kg+CORM-230mg/kg); and the Nrf2 knockout mice as before. The mice were killed after 4h and 16h of LPS administration, and liver and brain tissues were taken out. The expression of pro-inflammatory cytokines like TNF-a, IL-1β, IL-6 and iNOS in the liver and brain tissues were detected by RT-PCR; The expression of iNOS and ICAM-1 in the liver tissues were detected by western blot; and the pathology of liver and brain tissues were detected by HE staining. In mortality experiment,20 wild type and 20 Nrf2 knockout mice were provided respectively. The wild type mice were randomly divided into two groups:LPS group (LPS,10mg/kg); CORM-2 intervention group (LPS 10mg/kg+CORM-2 30mg/kg); and the Nrf2 knockout mice as before. Recording the mice number of surviving every 6h until 48h after LPS treatment.Results:The expression of pro-inflammatory cytokines like IL-1β, IL-6 and iNOS of liver tissues in the control group and the CORM-2 group of the wild type and the Nrf2 knockout mice were low. The expression of pro-inflammatory cytokines like IL-1β, IL-6 and iNOS at mRNA levels were substantially reduced after CORM-2 treatment (P<0.05); and the expression of iNOS and ICAM-1 at protein levels were reduced (P<0.05); and the neutrophile granulocyte infiltration in the liver tissues also reduced in the wild type mice, but not in the Nrf2 knockout mice. Similar to previous results, the expression of pro-inflammatory cytokines like TNF-α, IL-1β and IL-6 of brain tissues in the control group and the CORM-2 group of the wild type and the Nrf2 knockout mice were low. The expression of pro-inflammatory cytokines like TNF-a, IL-1β and IL-6 reduced after CORM-2 treatment in the wild type mice(P<0.05); and the neutrophile granulocyte infiltration in the brain tissues also reduced in the wild type mice, but not in the Nrf2 knockout mice. In mortality experiment, the wild type mice all died within 36h after LPS treatment, and the mortality of CORM-2 intervention group significantly reduced(P<0.05), but CORM-2 did not improve the mortality of the Nrf2 knockout mice.Conclusion:CORM-2 significantly reduced the expression of pro-inflammatory cytokines and inflammatory cell infiltration in the wild type mice, but not in the Nrf2 knockout mice. And CORM-2 significantly improved the mortality of the wild type mice but not the Nrf2 knockout mice. Therefore, the Nrf2 pathway play a vital role in carbon monoxide’s anti-inflammatory effect. |
PDF Full Text Request