Mechanism Research For Platycodin D Ameliorating Mouse Intestinal Inflammation Through AMPK-regulated Macrophage Polarization

Inflammatory bowel disease(IBD)refers to a range of chronic idiopathic conditions that occur in the gastrointestinal tract,including ulcerative colitis(UC)and Crohn's disease(CD),which can both cause severe diarrhea,pain,fatigue and weight loss.Due to its long course,difficulty to cure,high cost,poor control,easy deterioration and even disability,IBD has become a huge burden on human survival and life.Therefore,the development of safe and efficient new treatment drugs and treatment strategies for IBD is of great significance to the control and prevention of IBD.As an important component of the innate immune system,macrophages play an important role in the maintenance of intestinal homeostasis and are considered to be a new potential target for the treatment of IBD.Activated macrophages can be broadly divided into classically activated macrophages(M1)and alternately activated macrophages(M2).In the steady state,colonic macrophages exhibit an M2-like phenotype which is anti-inflammatory and pro-tolerant.In IBD patients,the increase in the number of M1 type pro-inflammatory macrophages can cause excessive production of pro-inflammatory cytokines,leading to immune imbalance and intestinal barrier damage,and promoting the development of intestinal inflammation.And M2 type macrophages have been shown to relieve intestinal inflammation and promote tissue damage repair.Therefore,drugs with the potential to regulate the polarization of macrophages may have a therapeutic effect on the occurrence and development of IBD.The activation of adenosine monophosphate-activated protein kinase(AMPK)can effectively inhibit the polarization of M1 type macrophages and drive the macrophages to change to the M2 phenotype.Studies have shown that AMPK agonists can regulate the immune response in IBD animal models and improve the symptoms.Platycodin D(PLD)is a triterpene saponin isolated from the roots of Platycodon grandiflorum.It has been shown to have various pharmacological activities such as anti-inflammatory,anticancer,anti-oxidation and anti-virus,but whether PLD can improve the intestinal inflammation has not been reported.Recent studies have shown that PLD has been found to show ability to activate AMPK in Hep G2 cells,brown adipocytes and other cells.However,it is not clear whether PLD can regulate the inflammatory response of macrophages through activating AMPK.Therefore,from the perspective of macrophage immune regulation,this study investigated the effect of PLD on alleviating intestinal inflammation and protecting intestinal function and its related mechanisms,and hoping to provide a theoretical foundation for the development of new IBD treatment drugs and treatment strategies.I.Effect of platycodin D on DSS induced colitis.Methods: DSS-induced colitis mice were established by giving 3%DSS drinking water for 7 consecutive days.Mice were treated with PLD for 7 days in advance until the end of the experiment on the 14 th day to observe the therapeutic effect.Body weight,stool consistency and rectal bleeding of mice were recorded daily.Disease severity of mice was evaluated by disease activity index(DAI).Colon length was measured and histopathological lesions were detected by HE staining.The expression levels of TNF-?,IL-6,IL-1? and IL-10 in serum were detected by ELISA.The mRNA levels of cytokines TNF-?,IL-6,IL-1? and IL-10,as well as the mRNA levels of tight junction proteins TJP1 and OCLN in colon tissues of mice were detected by RT-PCR.Intestinal permeability of mice was detected by FITC dextran method.The protein expression levels of tight junction proteins TJP1 and OCLN in mouse colon tissues were detected by immunohistochemistry.Results: DSS induced colitis mice showed weight loss,increase of DAI index,damage of colonic mucosa,decrease of crypts and infiltration of inflammatory cells in colon.The administration of platycodin D effectively alleviated the weight loss and DAI index induced by DSS in mice,increased the length of colon,effectively inhibited the damage of colon mucosa,increased the number of colonic crypts,and reduced the infiltration of inflammatory cells in colon tissue.DSS-induced colitis mice showed increased levels of inflammatory cytokines in peripheral blood and colon tissues,and PLD effectively inhibited the levels of inflammation in peripheral and colon tissues.DSS-induced colitis mice showed decreased mRNA and protein levels of tight junction protein,accompanied with increased intestinal permeability.PLD treatment effectively increased the mRNA and protein expression levels of tight junction protein,decreased intestinal permeability,and protected the intestinal barrier function of mice.II.Effect of macrophages on PLD treatment of DSS induced colitis.Methods: DSS mouse model was constructed and PLD was administered as described above.During the experiment,the macrophages were removed by intraperitoneal injection of clodronate liposomes.The proportions of macrophages in colon,peritoneal fluid,mesenteric lymph nodes and spleen of mice were determined by flow cytometry.Body weight,stool consistency and rectal bleeding of mice were recorded daily.Disease severity of mice was evaluated by DAI.Colon length was measured and histopathological lesions were detected by HE staining.Results: Intraperitoneal injection of clodronate liposomes effectively cleared the macrophages in colon,peritoneal fluid,mesenteric lymph nodes and spleen of DSSinduced colitis mice.DSS-induced colitis mice with macrophage depletion showed weight loss,increased DAI index,damage to the colonic mucosa,reduced number of crypts,and infiltration of inflammatory cells in colon.In the macrophage depletion groups,PLD could not alleviate the weight loss and DAI index induced by DSS,and had no significant improvement on the colonic length and histopathological changes of colon tissue.III.Effect of platycodin D on regulation of macrophage polarization and thepreliminary mechanism study.Methods: DSS induced colitis mouse model was prepared and PLD was administered.Flow cytometry was used to detect the proportion of macrophages and X the proportion of M1-type,M2-type macrophages in colon tissue and peritoneal lavage fluid of mice.RT-PCR was used to detect the mRNA levels of M1 type macrophage polarization markers iNOS,CD86 and M2 type macrophage polarization markers Arg1,CD206.Western Blot was used to detect the protein expression levels of iNOS and Arg1 in colonic tissues and the protein expression levels of PI3K/Akt and NF-?B pathways which related to macrophage polarization regulation pathway in peritoneal macrophages.LPS-stimulated RAW 264.7 macrophage inflammation model was constructed,and the expression levels of cytokines TNF-?,IL-6,IL-1? and IL-10 in the culture supernatant of RAW 264.7 cells were detected by ELISA.RT-PCR was used to detect the mRNA levels of cytokines TNF-?,IL-6,IL-1? and IL-10 and the mRNA levels of polarization markers iNOS,CD86,Arg1 and CD206 in RAW 264.7 cells.Immunofluorescence was used to observe the protein expression of iNOS and Arg1 in RAW 264.7 cells.Flow cytometry was used to detect the proportion of M1 and M2 macrophages in RAW 264.7 cells.Western Blot was used to detect the expression levels of iNOS and Arg1 and expression levels of proteins of macrophage polarization related PI3K/Akt and NF-?B pathway in RAW 264.7 cells.And nuclear translocation of NF-?B p65 was detected by Western Blot and immunofluorescence.Results: In the in vivo model,PLD administration decreased the number of macrophages in colon tissue and peritoneal lavage fluid,as well as decreased the proportion of M1 macrophages and increased the proportion of M2 macrophages.PLD administration inhibited the expression of iNOS and promoted the expression of Arg1 in colon tissue.PLD administration promoted the activation of the M2 macrophage polarization-related PI3K/Akt pathway and inhibited the activation of the M1 macrophage polarization-related NF-?B pathway in mouse peritoneal macrophages.In the in vitro model,PLD administration inhibited the inflammatory response of LPSstimulated RAW 264.7 cells,inhibited the expression of iNOS,reduced the proportion of M1 macrophages,promoted the expression of Arg1,and increased the proportion M2 macrophages.PLD administration promoted the activation of the M2 macrophage polarization-related PI3K/Akt pathway and inhibited the activation of the M1 macrophage polarization-related NF-?B pathway in LPS-stimulated RAW 264.7 cells.Meanwhile,the nuclear translocation of NF-?B p65 was also inhibited by PLD.IV.PLD regulated macrophage polarization by activating AMPK.Methods: DSS induced colitis mouse model was prepared and PLD was administered.Peritoneal macrophages from different groups of mice were isolated.The expression levels of AMPK and p-AMPK in peritoneal macrophages were detected by Western Blot.LPS-stimulated RAW264.7 cell model was constructed and the expression levels of AMPK and p-AMPK in RAW264.7 cells were detected by Western Blot.After the expression of RAW264.7 AMPK was silencing by siRNA,the protein expression levels of polarization marker iNOS,Arg1 and the proteins of macrophage polarization related PI3K/Akt and NF-?B pathways were detected by Western Blot.Nuclear translocation of NF-?B p65 was detected by Western Blot and immunofluorescence.Results: Both in vivo and in vitro,PLD administration effectively promoted the activation of AMPK in macrophages.After AMPK was silenced in the in vitro model,the effects of PLD administration on the protein expression levels of macrophage polarization markers iNOS,Arg1,the proteins of macrophage polarization related PI3K/Akt and NF-?B pathways and the inhibition of p65 nuclear translocation in LPSstimulated RAW264.7 cells were weakened.V.PLD protects the intestinal barrier function by activating AMPK.Methods: LPS-stimulated co-culture intestinal barrier model of RAW264.7 cells with Caco-2 cells were constructed and PLD was administered.The trans-epithelial electrical resistance of Caco-2 cells was measured by cell resistance meter.The expression levels of tight junction proteins TJP1 and OCLN in Caco-2 cells were detected by Western Blot.After treatment with AMPK inhibitor Compound C,the expression levels of tight junction proteins TJP1 and OCLN in Caco-2 cells were detected by Western Blot.XIIResults: The treatment of PLD effectively increased the trans-epithelial electrical resistance of Caco-2 cells in the co-cultured intestinal barrier model,and increased the expression levels of tight junction proteins TJP1 and OCLN.When AMPK inhibitor Compound C was used to inhibit the activation of AMPK in the co-culture system,PLD treatment could not effectively increase the protein expression levels of tight junction proteins TJP1 and OCLN in Caco-2 cells.Conclusion:1.PLD has a protective effect on DSS-induced colitis mouse model.2.Both in DSS induced colitis mouse model and in LPS stimulated RAW 264.7 cell model,PLD inhibited the M1 macrophage polarization and promoted the M2 macrophage polarization.3.PLD may regulate PI3K/Akt and NF-?B pathways through activating AMPK.4.PLD may protect intestinal barrier function and improve intestinal inflammation by regulating macrophage polarization via activating AMPK.Innovation:1.This study revealed that the protective effect of PLD on IBD mouse model.2.This study demonstrated that macrophages play an important role in the intestinal protection process of PLD in IBD mouse model by selective depletion of macrophages.3.Using in vivo and in vitro models,this study revealed that the function of PLD to inhibit M1 macrophage polarization and promote M2 macrophage polarization.And further clarify the molecular mechanism of PLD in improving intestinal inflammation and protecting intestinal barrier in mice by regulating macrophage polarization via activating AMPK.4.This study supplemented the development value of PLD as an anti-inflammatory drug,and provided theoretical foundation for the development of effective immunomodulators and novel natural drugs for IBD.