Based On The EPO Signal Pathway To Explore The Effect And Mechanism Of Salidroside In Inhibiting Bacterial Inflammation

ObjectiveBacterial inflammation is an urgent health problem,the most effective treatment of drugs is antibiotics,but in view of the rise of global anti-chemical bacteria and the lack of anti-drug new drugs,there is an urgent need to explore new prevention and control strategies.The pathogenic microbial invasion can induce an acute inflammatory response.At this time,the host automatically activates inflammation,which suggests that we can treat bacterial inflammation by promoting adhesion.In autistic inflammation,the anti-inflammatory retreating molecules can inhibit inflammation,promote tissue regeneration,and enhance bacteria kill and remove the irreplaceable role.Therefore,excavating new inflammatory retreats is one of the important tasks of exploring new treatment strategies,which may provide assistive role for antibiotic therapy.Erythropoietin(EPO)is a glycoprotein that is known for the formation of erythrocytes.The proliferation and differentiation of the bone marrow in the bone marrow is combined with EPO receptor(EPOR).There is evidence that macrophages can also express the ability of EPOR,EPO and macrophages to enhance the ability to remove apoptotic cells in macrophages,promote inflammation of sterile peritonitis.However,whether EPO works in bacterial inflammation and its possible mechanisms remains to be explored.Red view is an anti-inflammatory effect,and studies have shown that redview is increased by EPO.Whether the redview can be used to play a role in inflammation in inflammation.Therefore,this topic we will explore these issues.MethodsChapter 2:Macrophage EPO Signaling Regulates E.coli-induced Inflammation Regression(1)Inject low-dose Escherichia coli into the abdominal cavity of mice,take samples at 0,6,12,24,48 and 72 hours,observe the total number of inflammatory cells in the peritoneal lavage fluid by light microscope cell count,flow cytometric detection of neutral The ratio of granulocytes and macrophages,the plate coating method is used to calculate the number of colonies,and the cytokine flow cytometry kit is used to detect the content of MCP-1,IL-6,TNF-?,IL-1?,IFN-?,and IL-10.The ELISA kit was used to detect the EPO content in the peritoneal fluid,and the flow cytometry was used to detect the expression of EPOR in the peritoneal inflammatory cell membrane and the expression of Jak2 in the cell.(2)E.coli was used to induce bacterial peritonitis in EPOR-C or EPOR-cKO mice,and samples were taken at 0,6,12,24,48 and 72 hours,and the total inflammatory cells in the peritoneal lavage fluid were observed by light microscope cell count Number,flow cytometric detection of the ratio of neutrophils and macrophages,use plate coating method to calculate the number of colonies,use cytokine flow cytometry kit to detect MCP-1,IL-6,TNF-?,IL-1?,IFN-y,IL-10.(3)In low-dose Escherichia coli-induced peritonitis,rhEPO or PBS was given,and the samples were taken at 0,6,12,24,48,and 72 hours respectively.The light microscope cell count was used to observe the total number of inflammatory cells in the peritoneal lavage fluid.Detect neutral,cell and macrophage ratio,use plate coating method to calculate the number of colonies,cytokine flow cytometry kit to detect MCP-1,IL-6,TNF-?,IL-1?,IFN-?,IL-10.Chapter 3:EPO promotes macrophages to engulf E.coli in a low-inflammatory manner through CD36 induced by PPARy(1)Co-incubate Escherichia coli marked by baclight with primary macrophages,use flow cytometry to detect the effect of macrophages phagocytosis of Escherichia coli,use CFSE to mark Escherichia coli and co-incubate with primary macrophages,fluorescence microscope Observe the effect of macrophages phagocytosis of E.coli.(2)Pretreat EPOR-C and EPOR-cKO macrophages with rhEPO or PBS for 12 hours,add heat-inactivated E.coli and incubate for 12 hours,then use flow cytometry to detect the culture supernatant inflammatory factor MCP-1,IL-6,TNF-?,IL-1?,IFN-y and IL-10 levels.(3)EPOR-C or EPOR-cKO mice were injected intraperitoneally with rhEPO(5000IU/Kg)or PBS at the same time as the injection of E.coli.After 24 hours,the materials were taken and the E.coli antibody was used to detect the phagocytic effect of macrophages on E.coli.(4)In low-dose Escherichia coli-induced peritonitis,flow cytometry was used to detect the expression of macrophages PPARy at 0,6,12,24,48,and 72 hours.(5)In vitro,use WT,EPOR-C or EPOR-cKO mouse macrophages,pretreat rhEPO or PBS,and incubate the inactivated E.coli with the primary macrophages,using flow cytometry Cytometry was used to detect the expression level of PPAR?.(6)Induce peritonitis in WT,EPOR-C or EPOR-cKO mice,give rhEPO or PBS treatment,and detect the mRNA or protein expression level of macrophage PPAR?.(7)PPAR?-C or PPARy-cKO mice were given intraperitoneal injection of Escherichia coli to induce peritonitis,the total number of inflammatory cells in the peritoneal lavage fluid was observed by light microscope cell count,and the proportion of neutrophils in the peritoneal lavage fluid was measured by flow cytometry,calculated by the plate coating method Colony count:EPOR-cKO mice were given intraperitoneal injection of Escherichia coli to induce peritonitis,and RSG treatment was given to detect the total cell count,neutrophils,colony count,and inflammatory factors in the peritoneal lavage fluid.(8)E.coli was used to induce peritonitis in EPOR-CKO,PPARy-C or PPARy-cKO mice,and treated with PBS,EPO or RSG,and flow cytometry was used to detect the phagocytosis of E.coli by macrophages.In vitro,culture EPOR-CKO,PPARy-C or PPARy-cKO mouse primary macrophages and treat them with PBS,EPO or RSG,then incubate the fluorescently labeled Escherichia coli with the cells,and detect by flow cytometry Phagocytosis of E.coli by macrophages.(9)In low-dose Escherichia coli-induced peritonitis,samples were taken at 0,6,12,24,48,and 72 hours,and the CD36 expression of macrophages was detected by flow cytometry.(10)In vitro,culture WT,EPOR-C,EPOR-cKO,PPARy-C or PPAR?-cKO mouse macrophages,pretreated with PBS,rhEPO,RSG,and then inactivated E.coli with the primary The macrophages were co-incubated,and the expression level of CD36 was detected by flow cytometry.(11)E.coli was used to induce peritonitis in WT,EPOR-C,EPOR-cKO,PPARy-C or PPARy-cKO mice,and treated with PBS,rhEPO or RSG to detect the expression level of macrophages CD3 6.(12)Cultivate primary peritoneal macrophages,treat them with EPO,specific CD36 blocking antibody or isotype control antibody,then incubate E.coli with the cells,and use flow cytometry to detect the phagocytosis of E.coli by macrophages.(13)WT mice were treated with E.coli to induce peritonitis,treated with EPO,CD36 specific blocking antibody or isotype control antibody,flow cytometry was used to detect the phagocytosis of E.coli by macrophages,the ratio of neutrophils,and bacterial colonies.Number and inflammatory factors.Chapter 4:EPO enhances the therapeutic effect of antibiotics on bacterial inflammation(1)High-dose Escherichia coli was used to induce peritonitis.Two hours after the administration of Escherichia coli,EPO,ciprofloxacin,and a combination of the two were administered separately to detect the total inflammatory cell count,the proportion of neutrophils,and the number of colonies;The lung tissue was stained with HE.(2)Culture EPOR-C macrophages or EPOR-cKO macrophages in vitro,pretreated with PBS,rhEPO or RSG,and add fluorescently labeled Staphylococcus aureus or methicillin-resistant Staphylococcus aureus,using flow cytometry The instrument detects phagocytosis.(3)Use Staphylococcus aureus or methicillin-resistant Staphylococcus aureus to prepare a mouse skin inflammation model,and give rhEPO or vancomycin alone or a combination of the two.After sampling,the total cell count was calculated under light microscope,and the proportion of neutrophils and the levels of inflammatory cytokines MCP-1,IL-6,TNF-?,IL-1?,IFN-y and IL-10 were detected by flow cytometry.The number of bacteria was counted by the dilution spreading plate method,and the infiltration of skin inflammatory cells was detected by HE staining.Chapter 5:Salidroside inhibits bacterial inflammation by regulating EPO signaling(1)Use PBS or salidroside to treat self-limiting peritonitis induced by low-dose Escherichia coli.The samples were taken at 4,12,24,and 48 hours,Calculate the total number of cells under a light microscope,and detect the proportion of neutrophils by flow cytometry.Flow cytometry was used to detect the levels of mouse peritonitis inflammatory factors MCP-1,IL-6,TNF-?,IL-1?,IFN-? and IL-10.(2)High-dose Escherichia coli induced regression and delayed inflammation,treated with salidroside or ciprofloxacin alone,or combined treatment with both,samples were taken 24 hours later,and the total cell number was calculated under light microscope;detected by flow cytometry.The proportion of neutrophils and the levels of MCP-1,IL-6,TNF-?,IL-1?,IFN-y and IL-10;the number of bacterial colonies was calculated by the dilution spreading plate method.(4)Use Staphylococcus aureus or methicillin-resistant Staphylococcus aureus to prepare a mouse skin inflammation model,and give salidroside or vancomycin alone or a combination of both.After sampling,the total cell count was calculated under light microscope,and the proportion of neutrophils and the levels of inflammatory cytokines MCP-1,IL-6,TNF-?,IL-1?,IFN-?and IL-10 were detected by flow cytometry.The number of bacteria was counted by the dilution coating method,and the infiltration of skin inflammatory cells was detected by HE staining.(5)Give mice intraperitoneal injection of PBS or salidroside.After 24 hours,the samples are taken.The EPO level in serum or peritoneal fluid is detected by ELISA,and the EPOR level of macrophages is detected by flow cytometry.(6)Culture WT macrophages in vitro,pretreated with PBS and salidroside for 48 hours,and incubated with fluorescently labeled Escherichia coli.Flow cytometry was used to detect the phagocytic effect of macrophages on Escherichia coli.ResultsChapter 2:In the self-limited E.coli mouse model,the EPO concentration was significantly higher than that of mice that were not vaccinated with E.coli.In addition,EPOR,which is present in macrophages instead of neutrophils,continues to accumulate before inflammation subsides.The only direct signal molecule downstream of EPOR,phosphorylated JAK2,also keeps increasing expression in synchrony with EPOR.EPOR-cKO mice experienced delayed regression after inoculation with E.coli,while exogenous EPO can promote the regression of bacterial inflammation.Chapter 3:EPO has no direct antibacterial activity.In vivo and in vitro experiments have shown that EPO increases the phagocytosis of E.coli by macrophages while inhibiting the expression of pro-inflammatory factors MCP-1,IL-6 and TNF-?.EPOR-cKO mice have reduced E.coli phagocytic ability and increased expression of pro-inflammatory factors MCP-1,IL-6 and TNF-?.During the removal of E.coli by macrophages,EPO induced the expression of PPARy in macrophages,and the mRNA and protein levels of PPARy in EPOR-cKO mice decreased significantly.The deletion of the EPOR gene of macrophages will reduce the uptake of E.coli,while the PPARy agonist RSG can restore the phagocytosis of E.coli by macrophages.The deletion of PPARy gene in macrophages will also reduce the phagocytic function of E.coli.During Escherichia coli-induced peritonitis,EPO regulates the expression of CD36 in macrophages through PPARy.CD36 specific blocking antibody can attenuate the phagocytosis of E.coli by macrophages with increased EPO.Chapter 4:In E.coli-induced delayed peritonitis,compared with ciprofloxacin alone,EPO combined with ciprofloxacin treatment reduced the number of neutrophils,E.coli colony counts,and MCP-1 in the peritoneal lavage fluid of mice.,IL-6 and TNF-? levels.EPO cannot directly kill Staphylococcus aureus,but PO can significantly enhance the phagocytosis of Staphylococcus aureus or methicillin-resistant Staphylococcus aureus by macrophages.The macrophages lacking the EPOR gene have reduced phagocytic ability of Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.The PPAR? agonist RSG can restore the ability of EPOR gene-deficient macrophages to engulf Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.In the back skin inflammation induced by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus,EPO and vancomycin were used in combination.Compared with EPO or vancomycin alone,the number of neutrophils and bacterial colonies,MCP-1 and TNF-? were significantly reduced.Chapter 5:Salidroside can promote the inflammation of Escherichia coli-induced peritonitis,and can inhibit high-dose Escherichia coli infection,Staphylococcus aureus infection and methicillin-resistant Staphylococcus aureus infection.Rhodiola glycoside can increase the EPO content in mice,the expression of EPOR protein in peritoneal macrophages and the phagocytic ability of macrophages to E.coli.Conclusion1.Macrophage EPO signal regulates the regression of inflammation induced by Escherichia coli.2.CD36 induced by PPARy by EPO promotes macrophages to engulf E.coli in a low-inflammatory manner.3.EPO enhances the therapeutic effect of antibiotics on bacterial inflammation.4.Salidroside inhibits bacterial inflammation by regulating EPO signal.