Oridonin Retards The Progression Of Acute Kidney Disease By Inhibiting The Expression Of Mincle

Objective:Acute kidney injury can be caused by short-term ischemia-reperfusion,also known as ischemia-reperfusion injury.It is the most common clinical complication,and there is no effective treatment at present.Oridonin is a traditional Chinese herbal medicine extracted from oridon,which can inhibit tumors,inflammation and fibrotic.However,it is unknown whether oridonin can effectively inhibit the acute kidney injury model constructed after ischemia-reperfusion surgery.This study further explores the therapeutic effect and mechanism of Oridonin on AKI mice through in vivo and in vitro experiments in mice.It is expected to provide research ideas for clinical drug development around acute kidney injury.Methods:1?In vivo experiments to observe the effects of Oridonin on AKI kidneys:Studies were performed in male SPF C57BL/6J?8 to 10 weeks;20²5 g;n=18?.Divided into sham operation group?control group?,IRI model group?IRI group?,Oridonin treatment group?OR group?,6 mice in each group.Adaptive feeding was performed for one week,and then bilateral renal arteries were ligated for 35 min to produce an IRI animal model.The OR group was orally administered with Oridonin,while the control group and the IRI group were administered with the same volume of DMSO solvent.The mice were sacrificed after modeling 3 days.Serum creatinine?SCr?and blood urea nitrogen?BUN?were detected.Renal structure and inflammatory cell infiltration were observed by H&E staining.The severity of renal interstitial matrix deposition was observed by PAS staining.Immunohistochemistry and Western blot were used to detect inflammatory markers?iNOS?MCP-1?IL-1??IL-6?TNF-??and AKT/NF-?B pathway.2?In vitro experiments to observe the effects of Oridonin on macrophage inflammation and its mechanisms.1)In vitro experiments to observe the effects of Oridonin on macrophages:mouse macrophage cell line?Raw264.7?was placed in a cell incubator at 5%CO₂,37?and sufficient humidity,and cultured in DMEM high-sugar medium containing 10%FBS.When cell growth reached 80%confluency,digestion was performed with 0.25 g/L trypsin and passaged for later use.The cells were divided into normal control group?control group?,model group?LPS group?,solvent control group?LPS+DMSO group?,Oridonin treatment group?LPS+5?M OR group?.After 24 hours of treatment,cell morphology was observed by inverted microscope.Real-time PCR,ELISA and immunofluorescence were used to observe the changes of secreted inflammatory factors.Western Blot was used to observe the expression level of AKT/NF-?B related protein.In order to further explore its mechanism of action and determine that Oridonin is dependent on Mincle to exert its anti-inflammatory effect,transfect the Mincle plasmid into cells.Real-time PCR and ELISA were used to detect the secretion of inflammatory factors.Western Blot was used to observe the changes of AKT.Results:1?In vivo experiments:1)Effects of Oridonin on renal function in mice:the results of serum creatinine?SCr?and urea nitrogen?BUN?showed that compared with IRI model group mice,the treatment with Oridonin were significantly decreased,and the difference was statistically significant,indicating that Oridonin can slow down renal function damage.2)Renal pathology results:H&E and PAS pathological staining results showed that severe renal tubular dilatation,shed of renal tubular epithelial cells,and significant matrix protein deposition occurred in the IRI model group.After Oridonin treatment,renal tubular dilatation was significantly reduced,and the area of matrix protein deposition was reduced,indicating that Oridonin can reduce pathological changes.3)Effect on inflammatory factor secretion:Real-time PCR,ELISA and immunohistochemical results show that Oridonin can simultaneously reduce the secretion of inflammatory cytokines like IL-1??TNF-?and MCP-1 at the level of genes and proteins.4)Effect on Mincle expression:according to the results of immunofluorescence co-localization,the expression of macrophage surface marker F4/80 and Mincle was increased in the IRI model group and decreased after treatment.The differences were statistically significant.5)Effect of oridonin on AKT/NF-?B pathway related indexes:Western Blot results show changes in AKT/NF-?B related pathway indicators.This shows that the expression of AKT/NF-?B related signaling pathways is increased in the IRI model,and its expression is decreased after treatment,the difference is statistically significant.2?In vitro experiments:1)Effect on cell morphology:compared with the normal control group,the cells showed larger cell bodies,intracellular vacuoles,and cell morphology became very irregular in the model group.There was no significant difference in cell morphology between the solvent control group and the model group.After pharmacological intervention,the morphology recovered.2)Effect on proinflammatory cytokines:Real-time PCR,Western Blot and immunofluorescence results showed that the expression of inflammation-related proteins in the model group was significantly increased,and there was no significant difference in the expression of each indicator in the solvent control group and the model group.Intervention treatment with Oridonin can effectively reduce the expression of inflammation-related proteins.3)Effect on Mincle/AKT/NF-?B signal axis:Mincle/AKT/NF-?B significantly increased in gene and protein levels in the model group,and expression of Oridonin decreased after intervention.4)Cell recovery experiment:Compared with the group treated with Mincle plasmid and Oridonin alone,it was observed that the inflammatory factors secreted by the cells were significantly increased,and the expression of p-AKT was also significantly increased,indicating that Oridonin was anti-inflammatory.The effect depends on Mincle.Conclusion:1:Oridonin can partially restore the degree of acute kidney injury in mice with ischemia-reperfusion.2:Oridonin can reduce the expression of proinflammatory cytokines produced by Raw264.7 stimulated by LPS.3:Oridonin may exert anti-inflammatory effects by down-regulating Mincle expression and affecting the AKT/NF-?B signaling pathway.