Effect Of Isoliquiritigenin On Cisplatin-induced Acute Kidney Injury Through Formyl Peptide Receptor 2

Purpose:Acute kidney injury(AKI)is a very dangerous acute disease with high mortality,high morbidity and high medical costs.AKI temporarily lacks effective treatment measures,and the current treatment focus is to avoid the potential harm caused by nephrotoxic drugs.The research team found in the previous GEO gene database screening study that Isoliquiritigenin can regulate inflammation by regulating the expression of FPR2,and Isoliquiritigenin can relieve renal inflammation.This study intends to confirm through animal and cell experiments that Isoliquiritigenin improves inflammatory damage induced by cisplatin by inhibiting FPR2 protein expression.Methods:1.Animal experiments were used to observe the inhibitory effect of Isoliquiritigenin on cisplatin-induced kidney inflammation in AKI mice.Thirty-two male C57 males aged 6 to 8 weeks and weighing 20 to 22 g SPF were randomly divided into sham operation groups.(Sham group),CIS model group(AKI group),low-dose Isoliquiritigenin group 7.5 mg/kg,high-dose Isoliquiritigenin group 30 mg/kg,8 mice in each group.On the first day,mice were given 80 μL of cisplatin to make an AKI model.The Sham group was given an equal volume of normal saline for 3 consecutive days,once a day.The mice were sacrificed after 3 days.Serum was measured for serum creatinine(SCr),Urea nitrogen(BUN);renal histopathology observed changes in renal tubular morphology and inflammatory cell infiltration,renal interstitial matrix and collagen deposition.The inflammatory proteins FPR2,p-P65,IL-1β,IL-6,TNF-α were detected by immunohistochemistry(IHC)and Western blot.2.In vitro cell experiments to observe the effect of isoliquiritigenin on LPS-induced macrophage inflammation.Mouse bone marrow-derived macrophages(BMDM cells)were extracted and placed in a 5%C02 and 37 ℃ incubator with 10%FBS DMEM low-sugar culture medium and 30%L929 supernatant were pretreated for 5 days,0.25 g/L trypsin digested,and the cells were divided into control group(CTL group),model group(LPS group),and low-dose ISO Group(LPS+ISO 20 μM),high-dose group(LPS+ISO 40 μM),DMSO solvent control group,DMSO+LPS group.After the cells were stimulated with 200 ng/ml LPS,they were treated with Isoliquiritigenin(ISO)at 20 μM and 40 μM,respectively.After incubating for 24 hours,observe the morphological changes of the cells under a microscope;detect the expression of FPR2 and inflammatory factors p-P65,IL-1β,IL-6,TNF-α by immunofluorescence,Western blot,ELISA and PCR.Use the specific agonist of FPR2 to complete the cell recovery experiment,observe the role of FPR2 in regulating inflammation,and divide the cells into control group(CTL group),model group(LPS group),LPS+ISO 40 μM group,LPS+ISO 40 μM+MMK1 group Among them,MMK1 was set with three concentration gradients of 500 nM,1 μM,and 2 μM,and the cells were incubated for 48 hours.Western blot was used to detect the effect of MMK1 on the expression of agonistic proteins.At the same time,MMK1 was used to incubate cells at 1 μM concentration.The expressions of FPR2 and inflammatory factors p-P65,IL-1β,IL-6,TNF-α were detected by Western blot,ELISA and real-time quantitative PCR.Results 1.Animal experiments found that compared with cisplatin-induced AKI mice,the serum creatinine(SCr)and urea nitrogen(BUN)at low and high doses of Isoliquiritigenin intervention treatment decreased,and the high dose was more obvious.The pathological staining revealed that the kidneys of the model group had severe renal tubular dilatation and injury,severe renal tubular epithelial detachment,and tissue erosion.The damaged renal tubular area showed red-pink matrix protein deposition.Compared with rats,renal tubular dilatation and glycogen deposition were significantly reduced,indicating that isoliquiritigenin can significantly reduce the pathological damage of kidney in AKI mice.Western blot results showed that,compared with model animals,Isoliquiritigenin intervention can significantly reduce the expression of inflammatory factors FPR2,p-P65,IL-1β,IL-6 and TNF-αin mice.The macrophage cell recovery experiment found that compared with normal cells,the primary macrophages became larger and burr-shaped around the cells after 24 hours of LPS stimulation,and the cell state returned to normal after the intervention of Isoliquiritigenin;Western blot results showed After LPS stimulation,the expression of cellular inflammation-related proteins was significantly increased.The addition of Isoliquiritigenin,especially 40 μM intervention,could significantly reduce the expression of inflammatory proteins;compared with normal cells,the expression of FPR2 and inflammatory proteins in LPS-stimulated cells was significantly increased.After the intervention of Isoliquiritigenin,the expression in cells can be significantly reduced.After the intervention of FPR2 specific agonist MMK1,the effect of Isoliquiritigenin on inflammation disappeared,which could not effectively inhibit the expression of inflammatory proteins.Conclusions:1.ISO can protect renal damage and renal-function of cisplatin-induced AKI mice by inhibiting renal inflammation;2.ISO can reduce the expression of inflammatory proteins and FPR2 protein in primary macrophages induced by LPS;3.The molecular and cellular mechanism of ISO inhibiting renal inflammation is potenially achieved by regulating the FPR2 protein pathway.