Study On The Effect And Mechanism Of Rosmarinic Acid In The Treatment Of Hypertensive Nephropathy By Inhibiting The Activation Of NLRP3 Inflammasome

Objective:To explore the role of Nucleotide-binding oligomerization domain,Leucine rich Repeat and Pyrin domain containing 3（NLRP3）inflammasome in angiotensin II（Ang II）-induced hypertensive nephropathy,to observe whether rosmarinic acid can alleviate Ang II-induced hypertensive nephropathy,and to explore its possible mechanism.Methods:The model of hypertensive nephropathy in C57BL/6 mice（6～8 weeks）was established by subcutaneous pumping of Ang II,and the experimental groups were:Control group,Ang II group(1.46 mg·kg^-1·d^-1),Ang II+RA group(Ang II 1.46 mg·kg^-1·d^-1+RA 50mg·kg^-1·d^-1).10 mice in each group were intervened for 4 weeks.Blood pressure was detected by tail-sleeve method weekly.Urinary microalbumin,serum creatinine and blood urea nitrogen were detected by Nanjing Jiancheng detection kit.Renal macrophage infiltration was detected by immunofluorescence.The degree of renal fibrosis was detected by Masson staining.The NADP⁺/NADPH detection kit was applied to determined the activity of NADPH oxidase.The level of ROS was measured by dihydroethidium（DHE）of frozen sections.The expression levels of NADPH oxidase subunits p22phox,p47phox and NLRP3,ASC,Caspase-1,IL-1βand IL-18 were detected by real time fluorescence quantitative polymerase chain reaction（q-PCR）and western blotting.HK-2 cells were cultured in vitro,and the experimental groups were:Control group,RA group（50 mol/L）,H2O2 group（100μmol/L）,H2O2+RA group（H2O2 100μmol/L+RA 50μmol/L）.the changes of ROS were detected by fluorescence microscope,the expression of NLRP3inflammasome was detected by Western blotting,and the contents of IL-1βand IL-18 in the supernatant were detected by ELISA.Results:1.Increased expression of NLRP3 inflammasome in hypertensive nephropathy induced by angiotensin II.Compared with the control group,the Ang II group showed increased blood pressure,urinary microalbumin,serum creatinine and blood urea nitrogen,with statistically significant（P<0.05）.Immunofluorescence showed that the infiltration of inflammatory cells such as macrophages increased in Ang II group（P<0.05）.Masson staining revealed fibrotic lesions in the kidney of the Ang II group,which resulted in increased expression of Col-1andα-SMA（P<0.05）.Compared with the Control group,the NLRP3 inflammasome and its downstream inflammatory molecules such as IL-1βand IL-18 RNA and protein expression in kidney tissues increased in the Ang II group（P<0.05）.2.Rosmarinic acid attenuates hypertensive nephropathy by inhibiting the activation of NADPH oxidase/ROS/NLRP3 inflammasome pathway.Rosmarinic acid alleviated hypertensive nephropathy induced by Ang II significantly.Compared with Ang II group,rosmarinic acid reduced blood pressure,serum creatinine,blood urea nitrogen and urinary albumin excretion（P<0.05）.Macrophage infiltration was significantly decreased in Ang II+RA group showed in immunofluorescence（P<0.05）.Masson staining revealed that the degree of renal fibrosis was improved after rosmarinic acid treatment,and the expression of Col-1 andα-SMA decreased obviously（P<0.05）.further results showed that compared with Ang II group,the RNA and protein synthesis of NLRP3 inflammasome and its downstream inflammatory molecules IL-1βand IL-18decreased in Ang II+RA group（P<0.05）,and rosmarinic acid inhibited the synthesis and activity of NADPH oxidase and ROS（P<0.05）.3.Rosmarinic acid inhibits the expression of ROS and NLRP3 inflammaso-me-related molecules in HK-2 cells stimulated by H₂O₂.After H₂O₂stimulation,the expression of ROS,NLRP3,IL-1βand IL-18 increased,which was significantly different from the control group（P<0.05）.Rosmarinic can inhibite H₂O₂-induced NLRP3,IL-1β,IL-18 synthesis（P<0.05）.It is further proved that rosmarinic acid can improve hypertensive nephropathy by reducing the activation of NLRP3inflammasome at the cellular level.Conclusions:1.The expression of NLRP3 inflammasome is increased in hypertensive nephropathy.NLRP3 inflammasome play an important role in promoting renal inflammation and fibrosis and aggravating renal function damage in hypertensive nephropathy.2.Rosmarinic acid can alleviate hypertensive nephropathy induced by Ang II,which is mainly through the inhibition of NLRP3 inflammasome and its downstream IL-1βand IL-18.Further studies found that rosmarinic acid inhibited NLRP3 inflammasome mainly through the inhibition of NADPH oxidase,that is,rosmarinic acid attenuated hypertensive nephropathy by inhibiting NADPH oxidase/ROS/NLRP3 inflammasome pathway.