Preliminary Study On The Pharmacological Effects And Mechanism Of RDN Synergistically In The Treatment Of Sepsis

Objective: To investigate the pharmacological action and preliminary mechanism of Reduning(RDN)synergistic effect in the treatment of sepsis.Method: RDN pharmacodynamics experiments were performed in LPS stimulated RAW264.7 cells,mice intraperitoneal injection of LPS and rat CLP models.(1)The inhibitory effect of different concentrations of RDN solution on RAW264.7 cells were detected by CCK-8.The in vitro model of sepsis was performed by LPS to stimulate RAW264.7 cells.After modeling,different concentrations of RDN solution were added for co-culture for 12 h.The effects of RDN on the levels of TNF-α and IL-6 in the supernatant of LPS-stimulated RAW264.7 cells after 12 h of co-culture were detected by ELISA.(2)mice were injected intraperitoneally with 10 mg/kg LPS to make an vivo model of sepsis,and 64 Balb/c mice were randomly divided into 8groups,including Blank group,Model group,0.9% Saline group,RDN high dose group(21.38 g/kg),RDN medium dose group(10.69 g/kg),RDN low dose group(5.35 g/kg),Positive control group(Hydrocortisone Sodium Succinate 61.65 mg/kg)and Combination group(RDN 10.69 g/kg + HSS 61.65 mg/kg).30 min after modeling,the corresponding interventional drugs were injected into the tail vein.After 12 h,the mices were sacrificed by ocular arterial blood collection.The levels of serum TNF-α and IL-6 were detected by ELISA.(3)The rat sepsis model was prepared by Cecal Ligation and Perforation(CLP).126 male Wister rats were randomly divided into 9 groups,including Blank group,Model group,Sham group,0.9% Saline group,RDN high dose group(10.69 g/kg),RDN medium dose group(5.35 g/kg),RDN low dose group(2.67 g/kg),Positive control group(Cefameprazole Sodium 0.63g/kg)and Combination group(RDN 5.35 g/kg + CS 0.63 g/kg),After continuous modeling for 3 d,the rats were sacrificed by abdominal aorta blood collection.The survival rate was calculated at 72 h.The levels of serum TNF-α and IL-6 were detected by ELISA.AST,ALT,BUN and Cr were detected by automatic blood biochemical analyzer.The liver injury was observed by HE staining.The expression of TLR4 and NF-κBp65 protein was detected by immunohistochemistry.The expression of TLR4 and NF-κBp65 gene was detected by q PCR.Results:(1)The effect of RDN on the activity of RAW 264.7 cells showed that the cytotoxicity of RDN increases with increasing dose,and the injury of RDN to RAW264.7 cells was dose-dependent.The results of ELISA showed that RDN inhibited the release of TNF-α and IL-6 after LPS stimulation of RAW264.7 cells,showing a dose-dependent effect.(2)In the intraperitoneal injection of LPS model in mice,RDN combined with HSS can reduce serum TNF-α,IL-6 expression levels.(3)In the rat CLP model,RDN combined with CS reduced the mortality rate after CLP72 h,decreased the expression levels of serum TNF-α and IL-6,and improved liver and kidney.In addition,liver immunohistochemistry and q PCR results suggest that RDN combined with CS may reduce TLR4,NF-κBp65 protein and m RNA expression to improve sepsis inflammatory response in rats.Conclusions: RDN may inhibit the inflammatory response of sepsis through synergistic effect.Further studies in rat CLP model suggest that the mechanism may be related to TLR-4/NF-κB pathway.