Molecular Mechanism Of Oridonin Induced Apoptosis In Mouse Mesangial Cells Via Activation Of The ROS/NRF2/OSGIN1 Pathway

Objective:To investigate the effects of oridonin(ORI)on LPS-induced proliferation and apoptosis in rat mesangial cells and its possible molecular mechanisms,in order to provide new drug options for the treatment of mesangial proliferative glomerulonephritis.Methods:1.In vitro experiments:(1)Different concentrations of lipopolysaccharide(LPS)were applied to rat thylakoid cells,and the appropriate concentration and time of LPSinduced cell proliferation were determined by CCK-8 method to establish a thylakoid proliferative glomerulonephritis cell model.(2)ORI was added at appropriate concentrations of LPS as culture conditions,and the effects of ORI on LPS-induced proliferation of thylakoid cells were determined by Edu assay and CCK-8 method.(3)Flow cytometry was to detect the effect of ORI on apoptosis of thylakoid cells.(4)Transcriptome sequencing was to observe the effect of ORI on transcriptome signaling pathways in thylakoid cells.(5)To further explore the possible molecular mechanisms of the inhibition of proliferation and antioxidant capacity of ORI on thylakoid cells,the expression of apoptosis-related proteins B-cell Lymphoma 2(Bcl-2)and Bcl-2Associated X Protein(Bax)were detected by Western blot.)and nuclear factor(erythroid-derived 2)-like 2(NRF2),an oxidative stress-associated protein.(6)The intracellular ROS content was measured by immunofluorescence microscopy and immunofluorescence detector using reactive oxygen species assay kit to investigate the effect of ORI on the antioxidant capacity of LPS-induced proliferating thylakoid cells.(7)Western bolt,immunofluorescence assay and addition of ROS scavenger,NRF2 inhibitor ML385 to determine the relationship between OSGIN1,NRF2,and ROS.2.In vivo experiments: leave out Kunming healthy mice as a negative control group,the rest of the double hindfoot pads were injected subcutaneously with Freund’s complete adjuvant + BSA.thereafter,BSA was injected intraperitoneally for a total of4 weeks.The normal Kunming mice were used as the negative control group,and the rest were used as the model group,low concentration treatment group(10 mg/kg ORI)and high concentration treatment group(20 mg/kg ORI),respectively.The mice in each group were administered intraperitoneal injection of drug/saline once a day on day 0after modeling,and the treatment group was fed with the corresponding dose of ORI;the control group and the model group were given equal amounts of saline,and the drug was administered daily for 3 weeks.The mice were executed by spinal dislocation method one day after the completion of drug administration.The mice were routinely stained with HE,observed under a light microscope and photographed to detect the toxic effects of the drug on the heart,lung and liver,which were the major organs of the animals;after the treatment,the kidney tissues were divided into several parts,one part of the kidney tissues was used for morphological examination,and the others were used for m RNA and protein extraction.Results:1.In vitro experiments:(1)The CCK-8 assay revealed that 30 μg/m L LPS action for 24 hours had a good induction effect on the proliferation of thylakoid cells.(2)In addition to 30 μg/m L LPS,ORI was added at different concentrations(3.13 μmol/L,6.25 μmol/L,12.5 μmol/L and 25 μmol/L,respectively),and ORI was found to have an inhibitory effect on LPS-induced proliferation of thylakoid cells in a drug concentration-dependent manner by Edu assay and CCK-8 assay.(3)Flow cytometry showed that 25 μmol/L ORI treatment for 24 h induced most of the thylakoid cells to apoptosis,and early apoptosis was the main cause.(4)Transcriptome sequencing revealed that ORI induced differential expression of many genes related to cell cycle arrest,proliferation and apoptosis,among which the differential expression of oxidative stress induced growth inhibitor 1(OSGIN1)was the most significant.(5)Western blot assay showed that ORI resulted in upregulation of Bax to Bcl-2 ratio and upregulation of oxidative stress-related protein NRF2 expression in thylakoid cells.(6)Immunofluorescence microscopy and immunofluorescence detector showed that the intracellular ROS content increased with the increase of ORI concentration.(7)Western blot detected that the expression of NRF2 and its downstream target gene HO-1 was upregulated with the increase of ORI concentration,and immunofluorescence showed that NRF2 entry into the nucleus was increased.Adding NRF2 inhibitor ML385 stimulation,Western Blot results showed that OSGIN1 was down-regulated with both NRF2 and NRF2 downstream target gene HO-1,which verified OSGIN1 as a NRF2 downstream gene.The previous results showed that ORI could significantly increase the level of ROS in thylakoid cells,and the effect was inhibited after intervention with ROS scavenger NAC,and it could reverse the result of ORI inhibiting thylakoid cell proliferation,while the expression of NRF2 and OSGIN1,which were induced to be up-regulated by ORI,was also down-regulated.2.Animal experiments showed that ORI could improve the pathological symptoms of chronic nephritis in mice,and had a protective effect on renal oxidative damage through activation of the NRF2-dependent OSGIN1 pathway.Conclusion:1.ORI inhibits LPS-induced mesangial cell proliferation and promotes its apoptosis.2.ORI mediates high expression of OSGIN1 to inhibit mesangial cell proliferation and promote its apoptosis.3.ORI reduces the degradation of transcription factor NRF2 and promotes its entry into the nucleus.4.ORI mediates its effect on NRF2 via ROS.5.ORI ameliorates the pathological manifestation of LPS-induced chronic nephritis in mice.6.ORI mediates the nephroprotective effect of NRF2-OSGIN1 pathway on chronic nephritis in mice.