DICER Regulates Macrophage Clearance Of Apoptotic Cells,the Mechanisms And Chinese Medicine Intervention

Research purpose and background:Dicer belongs to the RNase III endonuclease family.It is an indispensable key molecule in microRNA processing and plays an important role in the function of microRNA.Dicer participates in regulating the expression of various genes through the regulation of microRNA generation and function,and regulates the physiological and pathological processes such as growth,development,maturity,and aging of organisms.Abnormal clearance of apoptotic cells can lead to systemic autoimmune diseases and is related to the occurrence of cytokine storms.Macrophages play an important role in clearance of apoptotic cells.Macrophages clear apoptotic cells in time,which can prevent the release of potentially inflammatory and immunogenic contents and avoid the activation of autoantigen-specific T cells in the body.Autoimmune tolerance,causing autoimmune diseases.Therefore,the effective elimination of apoptotic cells plays a key role in maintaining the body’s immune homeostasis and avoiding the occurrence of chronic inflammation and autoimmune disease.Curcumin is a traditional Chinese medicine monomer,which can be extracted from a variety of traditional Chinese medicines,such as turmeric,curcuma,galangal,Luo Han Guo,etc.and has a variety of pharmacological activities.Numerous animal and human studies have shown that curcumin can affect a variety of immune cell functions and reduce the severity of various immune system-related diseases.For macrophages,curcumin induces M0 and M1 macrophages to polarize to the M2 phenotype and inhibits the inflammatory response.Dicer has been shown to participate in the regulation of macrophage functional polarization,and microRNAs can regulate the phagocytosis of macrophages by targeting different genes,which suggests that Dicer may be involved in regulating macrophages to eliminate apoptotic cells.Our research aims to observe the regulatory effect of Dicer on macrophage clearance of apoptotic cells and its effect on immune tolerance,and to explore the mechanism of macrophage regulation of apoptotic cell clearance and immune tolerance.TCM monomers are screened to regulate the expression of Dicer in macrophages,thereby providing new mechanisms and therapeutic targets for TCM to participate in the elimination of apoptotic cells and related immune functions.Methods:1.Detect mRNA and protein levels of Dicer after bone marrow-derived macrophages and peritoneal macrophages phagocytosed apoptotic cells by RT-PCR and Western blot;detect the effect of conditioned medium of apoptotic cells on the expression of Dicer in peritoneal macrophages;Cytochalasin D was used to inhibit the phagocytic process of peritoneal macrophages,and then the expression of Dicer on peritoneal macrophages was detected by apoptotic cells;the expression of Dicer after phagocytosis of apoptotic Jurkat cells was detected,and the expression of Dicer after apoptotic neutrophils;in vivo experiments The expression of Dicer in spleen macrophages was detected 24 hours after the apoptotic cells were injected into the tail vein,and the expression of Dicer in peritoneal macrophages was detected 12 hours after the apoptotic cells were injected intraperitoneally.To determine the effect of apoptotic cell clearance on Dicer expression in macrophages.2.Laser confocal observation of the differences between WT mice andDicer-cko(myeloid cell Dicer knockout)peritoneal macrophages phagocytosing apoptotic thymocytes;flow cytometry was used to detect bone marrow-derived macrophages from WT mice andDicer-cko mice The difference in phagocytosis of apoptotic thymocytes by peritoneal macrophages;in vivo experiments,the difference in phagocytosis of spleen macrophages after 2 hours of apoptotic cell injection in the tail vein was detected,and the difference in phagocytosis of peritoneal macrophages after 1 hour of intraperitoneal injection of apoptotic cells;After dexamethasone induces thymus apoptosis in mice,weigh the thymus mass,measure the thymus apoptosis rate and the proportion of CD68 macrophages by flow cytometry;measure the bone marrow of 10-week-old and 60-week-old WT mice andDicer-cko mice by flow cytometry And spleen neutrophil apoptosis rate;RT-PCR detection of inflammatory factor expression in peritoneal macrophages of WT mice andDicer-cko mice after phagocytosing apoptotic cells in vitro;flow cytometry detection of WT mice andDicer-cko mice Inflammatory factor content of peritoneal macrophages after phagocytosing apoptotic thymocytes in vivo.To clarify the effect of macrophage Dicer on phagocytosis of apoptotic cells3.Take 8-10 weeks old WT mice andDicer-cko mice,extract peritoneal macrophages 72 hours after injection of thioglycolic acid,adhere to the wall in a 6-well plate,and add apoptotic thymocytes 5 times the macrophages.After 24 hours of incubation,Trizol extracts RNA and submits it for RNA seq.The difference factor is greater than 1.5,and P<0.05 is the differential gene screening parameter.Gene Ontology enrichment analysis is used to observe the biological effect mechanism of Dicer molecules.Pathway is enriched by KEGG to observe Dicer’ s comprehensive influence on the physiological and pathological processes of the body.By searching the database and reading the literature,classify the function of the differential genes and draw a heat map;to understand the mechanism of macrophage Dicer regulating apoptosis clearance;4.Cultivate E.G7 OVA cells and induce apoptosis with cisplatin;peritoneal macrophages of WT mice andDicer-cko mice,add 5 times more apoptosis E.G7 OVA cells than macrophages,and detect phagocytosis by flow cytometry Difference;peritoneal macrophages of WT mice andDicer-cko mice were stimulated with 5 times the apoptosis of macrophages E.G7 OVA cells in the presence of LPS for 24 hours,washed three times with HBSS,and added CFSE labeled OT-2 CD4 T cells,flow cytometry to detect CD4 proliferation after 72 hours;peritoneal macrophages of WT mice andDicer-cko mice were stimulated with soluble OVA for 24 hours in the presence of LPS,washed three times with HBSS,and added CFSE labeled OT-2 CD4 T cells,flow cytometry was used to detect CD4 proliferation after 72 hours;WT mice andDicer-cko were injected with thioglycolic acid for 48 hours,and CFSE-labeled OT-2 CD4 T cells were injected into the tail vein,and apoptosis was injected intraperitoneally 24 hours later.G7 OVA cells,after 72 hours,flow cytometry of CD4 proliferation in the spleen of mice.To determine the effect of the macrophage Dicer on T cell response.5.ELISA detection of serum ANA,ADA of 10-week-old and 60-week-old WT mice andDicer-cko mice;Record the urine output of 60-week-old WT mice andDicer-cko for 24 hours,and detect the urine and serum by colorimetric method Urea nitrogen,urinary protein,and creatinine content;flow cytometric detection of serum cytokine content;immunohistochemical HE staining,observation of mouse kidney,lung,and skin inflammatory infiltration;immunofluorescence C3 staining observation of mouse kidney classic complement deposition;TUNEL staining was used to detect spleen cell apoptosis.To observe the autoimmune system ofDicer-cko aged rats.6.CCK8 detects the cell viability of quercetin,curcumin,berberine,resveratrol,glycyrrhizic acid,andrographolide,and GraphPad Prism calculates the EC50 concentration of different Chinese medicine monomers;stimulates peritoneal macrophages of WT mice according to the EC50 concentration 24 hours were examined by RT-PCR.Dicer was detected.Dicer that induced the most significant increase in Dicer induced by macrophages was selected.After peritoneal macrophages were stimulated,apoptotic thymocytes were added and phagocytosis was detected by flow cytometry.Based on this,Chinese medicine monomers that can regulate the macrophage Dicer and affect the phagocytosis of macrophages were screened.Results:1.Bone marrow-derived macrophages and peritoneal macrophages phagocytize apoptotic cells gradually increase with stimulation time;conditioned medium of apoptotic cells cannot stimulate changes in Dicer expression of peritoneal macrophages;inhibit the phagocytosis of macrophages,wither Dead thymus cells do not affect Dicer changes in peritoneal macrophages;Dicer mRNA expression increases after peritoneal macrophages engulf apoptotic Jurkat cells or apoptotic neutrophils;Dicer mRNA expression in spleen macrophages increases after tail vein injection of apoptotic thymus cells;Intraperitoneal injection of apoptotic thymus cells increased expression of Dicer mRNA in peritoneal macrophages.2.Laser confocal analysis showed that the proportion of peritoneal macrophages inDicer-cko mice decreased;flow cytometry detected bone marrow-derived macrophages inDicer-cko mice,and the phagocytic rate of peritoneal macrophages was lower than that of the WT control;apoptotic thymocytes were injected into the tail vein Spleen macrophage phagocytosis rate decreased;intraperitoneal injection of apoptotic thymocytes decreased peritoneal macrophage phagocytosis rate;after dexamethasone injection in mice,thymus weight and thymus apoptosis rate ofDicer-cko mice were higher than those of WT mice.The proportion of CD68 macrophages is basically the same;there is no significant difference in the rate of neutrophil apoptosis in bone marrow between 10-week-old and 60-week-old WT mice andDicer-cko mice,and the neutrophil apoptosis rate in the spleen ofDicer-cko mice is more The apoptosis rate increased with age;the expression of inflammatory factors in peritoneal macrophages of WT mice andDicer-cko mice after phagocytosing apoptotic thymocytes in vitro showed that TNF-α,IL-6,IL-1β secretion increased.After phagocytic apoptotic thymocytes were phagocytosed by peritoneal macrophages from WT mice andDicer-cko mice,the secretion of IFN y,TNF-α,IL-9,IL-13,and IL-22 from ILperitoneal macrophages ofDicer-cko mice increased,and IL increased.-21 secretion was lower than that in WT mouse macrophages.3.RNA seq results,screening for differential genes with a multiple of 1.5 and a P<0.05,yielding 208 differential genes,including 51 up-regulated genes and 157 down-regulated genes;Gene Ontology enrichment analysis and molecular function enrichment Set 12 GO items,cellular component enriched 18 GO items,and participate in biological process(biological process)enriched 27 GO items,a total of 57 GO terms.Through KEGG enrichment analysis,a total of 179 Pathways were enriched,which were mainly concentrated in human diseases,body systems,cellular processes,environmental information transmission,metabolism and other KEGG A-level Pathways.By searching the database and reading the literature,the biological functions of the differential genes were determined.It was found that the functions of the differential genes were mainly concentrated in the three major categories of regulation or participation in phagocytosis,inflammation,and metabolic physiological processes.4.WT mice andDicer-cko mice peritoneal macrophages engulfed E.G7 OVA cells,Dicer-cko phagocytosis rate was lower;WT mice andDicer-cko mice peritoneal macrophages engulfed apoptosis in the presence of LPS E.After G7 OVA cells,the OT-2 CD4 proliferation rate was higher in theDicer-cko group;after OT mice andDicer-cko mice peritoneal macrophages ingested soluble OVA in the presence of LPS,both OT-2 CD4 T cells appeared significantly Proliferation,but more obvious inDicer-cko group.WT mice andDicer-cko mice were transfused with OT-2 T cells and injected with apoptotic E.G7 OVA cells intraperitoneally.OT-2 CD4 T cells in the spleen were significantly proliferated,but theDicer-cko group was more significant.5.Serum ANA and ADA expression in 10-week-old and 60-week-old WT mice andDicer-cko mice showed pathological increase in only 60-week-oldDicer-cko mice;renal function test results showed that DICER-c KO at 60-week-old was small Renal dysfunction in rats;inflammatory factors IFN-γ,TNF-α,IL-5,and IL-9 in peripheral blood serum of DICER-c KO mice at 60 weeks of age were higher than those in WT group;immunohistochemical staining showed DICER-c Significant inflammatory infiltration in the kidneys,lungs,and skin of KO mice;immunofluorescent C3 staining showed that C3 complement deposition was significantly higher in kidneys of DICER-c KO mice at 60 weeks of age than WT controls;TUNEL stained spleen DICER-c KO mice Show more apoptotic cells.6.CCK8 detection EC50 concentration of Chinese medicine monomer on macrophages is curcumin 32uMOL,berberine 17uMOL,quercetin 88uMOL,andrographolide 29uMOL,resveratrol 138uMOL,glycyrrhizic acid 134uMOL.;According to the EC50 concentration,stimulating peritoneal macrophages of WT mice for 24 hours,curcumin-induced macrophage Dicer expression was most significantly up-regulated,and flow cytometry showed that Dicer was significantly up-regulated;The apoptotic thymocytes had a higher phagocytic rate than the blank control group,and had no effect on theDicer-cko group.Conclusion:1.The phagocytosis of apoptotic cells by macrophages can induce the Dicer expression increase;2.Deleted macrophage Dicer reduces its ability to clear apoptotic cells;3.Dicer can regulate the expression of multiple genes in macrophages,and regulate the clearance of apoptotic cells;4.Macrophage Dicer regulatie the immune response of apoptotic cell-associated autoantigens;5.Macrophage Dicer deficient mice develop systemic lupus erythematosus;6.Curcumin can promote the expression of Dicer in macrophages and increase macrophages clear apoptotic cells.