Study On The Effect And Mechanism Of Matrine On Macrophage Immune Regulation And Apoptosis

Background:Matrine(MAT)is a natural quinolazine alkaloid distributed in Matrine.As a single herbal drug,matrine has few toxic and side effects and high safety.Studies have found that MAT has anti-inflammatory,anti-tumor,antiviral,antibacterial and anti-allergic pharmacological effects,and is widely used in the treatment of allergic skin diseases,hepatitis and a variety of inflammatory diseases.Because of its great potential in clinical application,it has been regarded as a research hotspot by many scholars.In this paper,the anti-inflammatory and anti-apoptosis mechanisms of MAT on macrophages were discussed.Objectives:The aim of this study is to investigate the effects of MAT on THP-1 derived macrophages under inflammatory stimulation environment,and the mechanism of MAT inhibiting inflammatory response and protecting cells from apoptosis,so as to provide a reliable theoretical basis for the wider clinical application prospect of MAT and related mechanism research.Methods:1.CCK-8 method was used to detect the effect of different concentrations of MAT(0.1,0.25,0.5,1.0 and 2.0 mmol/L)on the viability of macrophages within the safe concentration range of drugs,and to determine the effective concentration of MAT.The effect of MAT on the viability of macrophages was observed after different time(3,6,12,24,48 h),and the optimal time of MAT was determined.2.To further verify the effect of MAT pretreatment on macrophage viability under LPS-simulated inflammatory stimulation.Neutral red staining solution and E.coli phagocytosis test were used to verify the effect of MAT on the phagocytosis of macrophages.WB and PCR were used to verify the effect of MAT on the expression of TNF-α,IL-6 and IL-1β induced by LPS in macrophages.ELISA kit was used to detect the effect of MAT pretreatment on the release of inflammatory factors TNF-α,IL-6 and transforming growth factor β1 secreted by macrophages under LPS stimulation.3.Hoechst staining and Caspase-3 activity detection reagent were used to observe the effect of MAT on the morphology of macrophages under inflammatory stimulation.Flow cytometry and Western blotting were used to detect the effect of MAT on LPS-induced macrophage apoptosis rate and the expression levels of apoptosis-related proteins Bax,Bcl-2,Cleaved-PARP and Cleaved-Caspase-3.4.Western blot was used to further verify the protein expression of TLR4/NF-κB signaling pathway related to LPS-induced inflammation.Immunocytofluorescence was used to observe the nuclear entry of nuclear transcription factor NF-κB under inflammatory stimulation and the changes of TLR4/NF-κB signaling pathway after MAT pretreatment.To explore the possible mechanism of MAT.5.Bioinformatics technology was used to identify the mirnas that interacted with TLR4,and to study whether they could interact with MAT.The expression of mi R-30 b was verified by PCR that THP-1 cells were induced to differentiate into macrophages in vitro and then treated with LPS or MAT.Western blotting was used to detect the expression levels of TNF-α,IL-6,IL-1β and apoptosis-related proteins Bax,Bcl-2,Cleaved-PARP and Cleaved-Caspase-3 after mi R-30 b was silenced.The effect of mi R-30 b silencing on TLR4/NF-κB signaling pathway was further verified.Results:1.The results of CCK-8 showed that MAT had no significant effect on the viability of macrophages when the concentration was 0.05-1.0 mmol/L(P>0.05).When the concentration was >1.0 mmol/L,the viability of macrophages was significantly inhibited,so 1.0mmol/L was selected as the concentration of MAT for subsequent experiments.The action time of MAT was time-dependent with the viability of macrophages,and the longer the action time,the more toxic the drug was to the cells.Therefore,24 h was selected as the best action time of MAT.2.After co-treatment of macrophages with LPS(1.0 μg/m L)and MAT(1.0mmol/L),the viability of macrophages increased with the pretreatment concentration of MAT in a dose-dependent manner.MAT pretreatment significantly increased the phagocytic capacity of macrophages,increased the expression of transforming growth factor β1,and inhibited the expression of inflammatory factors and inflammation-related proteins secreted by macrophages.This suggests that MAT can inhibit LPS induced inflammatory injury in macrophages.3.Hoechst staining and flow cytometry results showed that MAT intervention could significantly inhibit LPS-induced apoptosis of macrophages,not only reducing the activity of Caspase-3 induced by LPS,but also reducing the activity of caspase-3induced by LPS.MAT also inhibited the expression of apoptosis-related proteins Bax,Bcl-2,Cleaved-PARP and Cleaved-Caspase-3,indicating that MAT could protect macrophages from LPS-induced apoptosis.4.In order to explore the specific mechanism of MAT inhibiting inflammatory response and anti-apoptosis,the expression of TLR4/NF-κB,a classical inflammatory pathway,was detected by related techniques.MAT treatment could inhibit the activation of TLR4/NF-κB signaling pathway induced by LPS and reduce the nuclear activation of NF-κB.These results indicate that MAT can inhibit the activation of NF-κB and thus produce anti-inflammatory and anti-apoptotic effects.5.Mi R-30 b plays an important role in the progression of cancer,while it is relatively less studied in the effects of MAT immunomodulation and anti-apoptosis.Bioinformatics analysis of the database predicts that mi R-30 b and TLR4 3’UTR base complementary pairing,indicating that it has a target for interaction.Compared with the control group,the expression level of mi R-30 b was significantly down-regulated.In LPS-simulated inflammatory cell model,mi R-30 b expression was also significantly decreased,and MAT treatment could reverse mi R-30 b expression.After silencing mi R-30 b,the expression levels of inflammatory factors TNF-α,IL-6,IL-1β and apoptosis-related proteins Bax,Bcl-2,Cleaved-PARP and Cleaved-Caspase-3 were significantly increased,and the expression levels of related pathway proteins were also reversed.This suggests that MAT may inhibit the activation of the signaling pathway TLR4/NF-κB by increasing the expression of mi R-30 b.Conclusion:This study confirmed that MAT may inhibit the activation of TLR4/NF-κB signaling pathway by increasing the expression level of mi R-30 b,and enhance the immunomodulatory effect of macrophages,thereby protecting macrophages from inflammatory injury and apoptosis.