Curcumin Inhibits Oxidative Stress Of Macrophages Infected By Mycobacterium Tuberculosis Based On Nrf2 Pathway

Objective: The problem of drug resistance of tuberculosis is becoming more and more serious,and the control and treatment of tuberculosis in China and even the whole world are still facing severe challenges.Therefore,it is of great significance to find new safe and effective anti-tuberculosis drugs.In this study,macrophages were infected with attenuated strain of Mycobacterium bovis(Mycobacterium bovis M-bovis)to establish an in vitro oxidative stress model,and to explore the inhibitory effect of curcumin(Curcumin;Cur)on oxidative stress of macrophages infected with Mycobacterium tuberculosis based on Nrf2 pathway,in order to provide some experimental basis for exploring the pathogenesis of tuberculosis and developing host-oriented therapy.Methods: The infection model was established with THP-1-derived macrophages.The experiment was divided into four groups:(1)Control group;(2)M-bovis group;(3)M-bovis+Cur group;(4)M-bovis+Cur+ML385 group.The cells were collected after 48 hours of culture according to the above grouping method.Reactive oxygen species(ROS)detection kit was used to observe the fluorescence intensity of ROS under fluorescence microscope,the content of reduced glutathione(GSH)was detected by colorimetry,the protein expression of Nrf2 and its downstream target genes NQO1 and HO-1 were detected by Western Blotting,and the cell proliferation rate of each group was detected by MTT method.,The macrophage load of each group was detected by plate counting method.Results:1.Toxicity of curcumin Low dose curcumin had no toxic effect on macrophages,but curcumin above 30 μM had certain toxicity to macrophages and inhibited the proliferation of macrophages in a dose-dependent manner(P < 0.01).2.ROS fluorescence intensity of macrophages in each group : Compared with Control group,ROS fluorescence intensity of macrophages in M-bovis group,M-bovis+Cur group and M-bovis+Cur+ML385 group increased significantly;compared with M-bovis group,ROS fluorescence intensity decreased significantly in M-bovis+Cur group;after intervention with Nrf2 inhibitor ML385,intracellular ROS fluorescence intensity increased again.3.GSH content of macrophages in each group:There were significant differences in GSH content among the three groups.Compared with Control group,GSH content in M-bovis group decreased significantly(2.93 ±1.20 vs 7.34 ±2.21,P < 0.01),and GSH content increased significantly after curcumin treatment(5.87 ±1.29 vs 2.93 ±1.20,P <0.05),while Nrf2 inhibitor ML385 reversed the above effects of curcumin.4.Expression of Nrf2 protein in macrophages of each group:There were significant differences in the expression of Nrf2 protein among the three groups.Compared with Control group,the expression of nuclear Nrf2 protein in M-bovis group decreased significantly,while the expression of Nrf2 protein in M-bovis+ Cur group was significantly higher than that in M-bovis group(P < 0.0l),and the expression of ML385,Nrf2 protein in cells treated with Nrf2 inhibitor Cur was significantly decreased(P<0.01).5.Expression of NQO1 and HO-1 proteins in macrophages of each group: There were significant differences in NQO1 and HO-1 protein expression among different groups(P <0.05).Compared with Control group,the protein expression of NQO1 and HO-1 of Nrf2 downstream gene in M-bovis group decreased significantly,while the protein expression of NQO1 and HO-1 in M-bovis+ Cur group was significantly higher than that in M-bovis group(P < 0.05),and Nrf2 inhibitor ML385 reversed the above effects of curcumin.6.Macrophage proliferation rate of each group: There were significant differences in macrophage proliferation rate among all groups(P < 0.05).Compared with Control group,the macrophage proliferation rate of M-bovis group decreased significantly,compared with M-bovis group,the macrophage proliferation rate of M-bovis+Cur group increased significantly,but the cell proliferation rate of M-bovis+Cur+ML385 group decreased again.7.The amount of bacteria carried by macrophages in each group : There were significant differences in the bacterial load of macrophages among all groups(P < 0.01).Compared with M-bovis group,After the cells were treated with curcumin,the bacterial load decreased significantly(P < 0.01).However,Nrf2 inhibitor ML385 was given,The cell bacterial load increased significantly in M-bovis+Cur+ML385 group.Conclusion: Curcumin can inhibit the oxidative stress of macrophages induced by Mycobacterium bovis by increasing the expression of Nrf2 and inducing the transcription of downstream antioxidant molecules,promote the proliferation rate of macrophages and enhance the clearance and killing of M-bovis by macrophages.