| Objective:The different.phenotypes of macrophages are activated by the microenvironment,which is called macrophage polarization.Macrophages are mainly activated into the M1 phenotype and the M2 phenotype,which play different functions and are associated with various diseases.The complicated mechanism linked to macrophage polarization remains to be discussed.Sinomenine is an alkaloids compound with various pharmacological actions such as anti-inflammatory,analgesic,anti-tumor and immunosuppression.Previous results showed that sinomenine inhibited classically macrophage activation induced by LPS and alternatively activated macrophages induced by IL-4.In vitro and in vivo experiments have shown that sinomenine regulates the expression of α 7nAChR,while α 7nAChR is an important target for sinomenine to exert anti-inflammatory effects.Based on this,this experiment will explore the relationship between sinomenine-mediated macrophage polarization and a 7nAChR.We firstly confirmed the effect of sinomenine on LPS-induced M1 macrophage polarization and α 7nAChR expression level,and analyzed the correlation between α 7nAChR expression and M1 polarization.Further we constructed a cell model of α 7nAChR gene silencing,and observed the effect of α 7nAChR gene silencing on M1 polarization.On the other hand,we compared with M2 macrophage polarization model induced by IL-4 and IL-4/6,and explored the effect of sinomenine on M2 polarization and α 7nAChR expression.Besides,we explored the effect of gene silencing on M2 polarization.From the above aspects,the effect of α7nAChR on macrophage polarization was analyzed and elucidated the mechanism of action of sinomenine on α 7nAChR regulation and macrophage polarization.Methods:1.The role of α 7nAChR in M1 polarization and the effect of Sinomenine1.1 Effects of sinomenine on M1 polarization and α 7nAChR expressionRAW 264.7 cells induced by LPS were treated by Sinomenine to analyze TNF-αlevels in cell supernatant by ELISA.The expression of iNOS and IL-6 mRNA were determined by RT-qPCR.Western blot was used to detect the expression level of α 7nAChR protein.1.2 Construction of α 7nAChR gene silencing RAW 264.7 cell lineIn this study,the recombinant lentivirus vetor was transfected into RAW 264.7 cells,which was used to silence α 7nAChR genes.After exploring the optimal conditions for lentiviral transfection,based on the antibiotic resistance of lentivirus vector,the dose of Puromycin was selectd to screen the cells which were silenced.The α 7nAChR levels wer e determined by Western Blot which was used to identify the effect of the α 7nAChR shRNA.1.3 Effect of α 7nAChR gene silencing on macrophage proliferationTo discuss the effect of α 7nAChR genes silencing on cell proliferation,CCK-8 assay was used to detect and compare with the absorbance values of the cells transfected with the negative control lentivirus and the lentivirus withα 7nAChR shRNA at 24h,48h,72h and 96h after seeding at different cell inoculation concentrations.1.4 Effect of α 7nAChR genes silencing on M1 macrophage polarizationELISA was used to detect TNF-α secretion levels of those two kinds of cells at different time points,and the effect of α 7nAChR genes silencing on the expression of inflammatory cytokines and its mannner were investigated.Besides,to elucidate the effect of α 7nAChR genes silencing on M1 polarization,those two kinds of cells treated with LPS for 24h and RT-qPCR was used to determine the expression of TNF-α、IL-6、iNOS mRNA.2.The role of α 7nAChR in M2 polarization and the effect of Sinomenine2.1 Exploring the time of IL-4 induced M2 macrophage polarization and the dose of sinomenine and nicotineThe effect of IL-4 on the secretion of IL-10 in RAW 264.7 cells at different time points was detected by ELISA.At the dose of 10ng/mL,IL-4 was treated at 12h,24h,36h,48h and determined the administration time.Subsequently,analyzed the different concentrations of sinomenine and nicotine on IL-10 secretion in RAW 264.7 cells induced by IL-4 were used to determine the dose of sinomenine and nicotine.2.2 The comparison of M2 macrophage polarization and the expression of α 7nAChR which the macropahes were stimulated by IL-4 and IL-4/6With treatment of IL-4,IL-6 and IL-4/6,analyzed the expression of Arg-1、Fizz1、Ym1 mRNA and IL-10 by RT-qPCR and ELISA respectively,to take comparison of these two M2-polarized models.Western Blot was used to detect the expression of α 7nAChR which the macrophages treated with IL-4 and IL-4/6,to discuss the relationship between α 7nAChR and M2 polarization preliminary.2.3 Effects of sinomenine on M2 polarization and α 7nAChR expression of macrophagesNicotine,as the positive control,and sinomenine were administered to explore the effects of drugs on M2 polarization by determining the expression level of Arg-1 and IL-10.And α 7nAChR levels were detected at the same time.These researches are to discuss the effect of sinomenine on M2 polarization and α 7nAChR expression,meanwhile,investgating the crosstalk betweenα 7nAChR and M2 polarization.2.4 Effect of α 7nAChR genes silencing on M1 macrophage polarizationTo investigate the relationship between α 7nAChR and M2 polarization,RT-qPCR was used to determine the levels of Arg-1、Fizz1、Ym1 mRNA which the Negative Control cells and α 7nAChR shRNA cells stimulated by IL-4/6.Results:1.The role of α 7nAChR in M1 polarization and the effect of Sinomenine1.1 Effects of sinomenine on M1 polarization and α 7nAChR expressionThe results showed that compared with the control group,the secretion of TNF-α and the expression of iNOS and IL-6 mRNA in the LPS group were significantly increased whereas compared with the LPS group,the above indicators were significantly down-regulated after the administration of sinomenine.Meanwhile,sinomenine significantly decreased the upregulation ofα 7nAChR induced by LPS.1.2 Construction of α 7nAChR gene silencing RAW 264.7 cell lineRAW 264.7 cells were transfected with lentivirus,with setting the conditions of MOI=200,100,50,20,and 10 and four different culture conditions:control group,conventional medium group,HitransG A group and HitransG P group,observe the fluorescence expression of 72 hours after transfection.Finally,it was determined that the MOI=100 combined with the HitransG P were used as the condition of lentivirus transfection.The proliferation of different concentrations of puromycin was detected by CCK-8 assay.The OD values at 24h,48h and 72h after administration were observed.The cell viability of the 2 μg/mL puromycin group was 5.02%at 48h.The cell survival rate at 72 h was 3.15%;in the 5 μg/mL group of puromycin,the cell viability at 48 h was 1.80%,and that was 0.56%at 72h.Therefore,2 μg/mL of puromycin was selected as the initial screening stable strain in the subsequent experiments.The expression of α 7nAChR in α 7nAChR gene-silencing cells was determined by Western Blot.The results showed that the α 7nAChR expression level ofα 7nAChR gene silencing group was inhibited compared with the negative control group,suggesting that the cell model of α 7nAChR gene silencing was successful ly constructed.1.3 Effect of silencing α 7nAChR gene on macrophage proliferationCell proliferation was detected by CCK-8 assay at 24h,48h,72h and 96h between the negative control group and α 7nAChR gene silencing group at different cell concentrations.The results showed that when the cells were in the logarithmic growth phase,the cell survival rate of the negative control group was significantly higher than that of the silencing group.1.4 Effect of α 7nAChR genes silencing on M1 macrophage polarizationThe TNF-α secretion of α 7nAChR gene silencing cells at different time points with LPS administration was detected by ELISA.The results showed that TNF-α secretion of LPS group in α 7nAChR shRNA cell was significantly decreased compared with LPS group of negative control group.RT-qPCR was used to detect the levels of TNF-α、IL-6、iNOS mRNA which the Negative Control cells and α 7nAChR shRNA cells were treated with LPS for 24h.It showed that compared with the Negative Control cells stimulated by LPS,the levels of TNF-α、IL-6、iNOS mRNA which the α 7nAChR shRNA cells were stimulated by LPS were significantly decreased.2.The role of α 7nAChR in M2 polarization and the effect of sinomenine2.1 Exploring the time of IL-4 induced M2 macrophage polarization and the dose of sinomenine and nicotineThe effect of IL-4 on IL-10 secretion of RAW 264.7 cells at different time points was determined by ELISA.The dose of IL-4 was determined to be 10 ng/mL,and the administration time was 24 h.IL-10 secretion of RAW 264.7 cells induced by IL-4 with the administration of sinomenine and nicotine was detected by ELISA.The results showed that compared with the IL-4 group,IL-10 release was significantly inhibited in the groups which the doses of sinomenine were 200 μ M,400 μ M,800 μ M and 1000 μ M.And it was in a dose-dependent manner.In the subsequent experiments,the concentration of sinomenine will be 400 μM.Meanwhile,nicotine can significantly inhibit the secretion of IL-10 at the dose of 400 μ M,so the subsequent dose of nicotine was 400 μ M.2.2 The comparison of M2 macrophage polarization and the expression of α 7nAChR which the macropahes were stimulated by IL-4 and IL-4/6To investigate the effects of M2 polarization induced by IL-4 and IL-4/6,IL-10 release and Arg-1 mRNA expression were detected by ELISA and RT-qPCR respectively.Those of IL-4/6 group were significantly higher than IL-4 group.Western Blot was used to detect the expression of α 7nAChR which the macrophages were treated with IL-4 and IL-4/6.It showed that compared with control group,there was no significant difference in the expression level of a 7nAChR protein in the IL-4 group,while the expression level of α 7nAChR protein in the IL-4/6 group was significantly up-regulated.Compared with the IL-4 group,the IL-4/6 group was compared with the IL-4/6 group.The expression level of α 7nAChR protein was significantly up-regulated.2.3 Effects of sinomenine on M2 polarization and α 7nAChR expression of macrophagesThe results showed that sinomenine or nicotine could significantly down-regulate the expression of IL-10,Arg-1,Fizzl and Ym1 induced by IL-4 and IL-4/6.At the same time,the expression level of α 7nAChR was investigated.The results showed that the expression level of α 7nAChR was significantly increased in IL-4/6 group compared with the control group.With the administration of sinomenine or nicotine,the expression of α 7nAChR was significantly down-regulated compared with IL-4/6 group.2.4 Effect of α 7nAChR genes silencing on M1 macrophage polarizationRT-qPCR was used to determine the levels of Arg-1、Fizz1、Ym1 mRNA which the Negative Control cells and α 7nAChR shRNA cells stimulated by IL-4/6.It showed that the expression of Arg-1、Fizz1、Ym1 mRNA in IL-4/6 group of α 7nAChR shRNA cells was significantly up-regulated compared with IL-4/6 group of Negative Control cells.Conclusion:1.Sinomenine can inhibit the M1 polarization and decrease the expression ofα 7nAChR.2.α 7nAChR involves in M1 polarization;the deletion of α 7nAChR can inhibit the M1 polarization of macrophages,suggesting that both sinomenine and nicotine can inhibit the M1 polarization by down-regulating α 7nAChR3.Sinomenine can inhibit the M2 polarization.4.There is no difference on α 7nAChR levels between normal macrophages and macrophages treated with single IL-4.However,the expression of α 7nAChR is significantly increased by combined IL-4/IL-6 treatments;and it is downregulated after sinomenine administration.5.α 7nAChR deficiency can promote the M2 polarization and the mechanism of sinomenine is complicated on regulating M2 polarization.6.Sinomenine modulates M1 polarization by regulating α 7nAChR levels.Down-regulating the levels of α 7nAChR can inhibit M1 polarization and promote its transition to M2 phenotype. |
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