Protective Mechanism Of Lidanpaidu Prescription On LPS-Induced Acute Kidney Injury In Mice And Forming Technique Of Its Granule

The Lidanpaidu Prescription(LDP),a hospital preparation,composed of Chinese classical preparations,has been reported to have antiendotoxin,anticoagulant,improving immunity and other effects.Sepsis is a systemic inflammatory response syndrome(SIRS)caused by infection.Acute kidney injury(AKI)caused by sepsis is one of the common causes of death in ICU patients.However,there is still no good treatment for acute renal injury caused by sepsis.For decades,the anti-inflammatory drug Glucocorticoid(GC)is used for adjuvant treatment of sepsis.There have been disputes over the use of dosages and so on.Therefore,we pretreated mice with different doses of LDP to explore the protective effect and mechanism in AKI caused by Lipopolysaccharide(LPS)in mice.Objective: To explore the protective effect of LDP on the survival rate of LPS-induced sepsis in mice.To explore the protective effect and the possible molecular mechanism of action of LDP in AKI caused by LPS in mice,and to explore the suitable dosage forms for clinical.Methods: a lethal sepsis model was made by intraperitoneal injection of LPS.60 Balb/c male mice were randomly divided into four groups: control group,model group,LDP group and dexamethasone group.The reaction and death of mice were observed every 4 h within 48 h after injection LPS.In the second round of studies,48 mice were randomly divided into six groups(n=8 each group): control group,model group(LPS 7 mg/kg),LDP(11.9,23.8,47.6 g/kg)groups,and dexamethasone hydrochloride(5 mg/kg)group.All the mice were dosed by intragastric administration for seven days.One hour after the last intragastric administration,the model group,LDP group and dexamethasone group were injected intraperitoneally with LPS(7 mg/kg).Six hours after the LPS injection,the mice were sacrificed and the blood and kidney tissues were collected at the peak of kidney injury according to the literature and preliminary experiment.The kidney injury induced by LPS was assessed by histological examination.The levels of TNF-?,IL-6,IL-10 and other inflammatory factors in serum and kidney homogenate were detected by Enzyme linked immunosorbent assay(ELISA).The expression of the inflammatory genes TNF-? and IKK? mRNA was detected by RT-PCR.Western blot was used to detect the expression of phosphorylated NF-?B in cytoplasm and nucleus and the expression of I?B,phosphorylated I?B.The expression and localization of nuclear factor NF-?B p65 protein were detected by laser confocal immunofluorescence.All these experiments were to observe the the protective effect of LDP and to explore the effect of LDP on activation of NF-?B signaling pathway.Using the ratio of briquetting,moisture absorption rate and solubility rate as the reference index to screen the excipient of LDP.Screen the best granulating process with drug-excipient ratio,excipient ratio and ethanol concentration as indexes,and measure the relative critical humidity.Results: 1.the survival test: All mice in each group were delayed and sluggish after injection LPS.They trembled all over and did not drink and eat.Their hair was shiny and shuddered.The survival rate of mice in 48 hours in the LDP group was 40% higher than that in the model group,and the dexamethasone group's was 53.3% higher than that in the model group.The results showed that LDP can effectively improve the survival rate of the mice with fatal sepsis.2.HE staining sections observed pathological damage: there were obvious injuries in the kidney tissues in the LPS group,including flat and sloughed tubular epithelial cells,loss of the brush border and visible bare membranes.These kidney tissue injuries were markedly ameliorated in the LDP(11.9,23.8,47.6 g/kg)and dexamethasone(5 mg/kg)groups.3.Inflammatory factors in serum and kidney: There was a dramatic increase in inflammatory cytokine expression in the model group.The inflammatory factor levels in LDP(11.9,23.8,47.6 g/kg)groups and dexamethasone hydrochloride(5 mg/kg)group were significantly decreased(P<0.01),in a dose-dependent manner.4.Detection of TNF-? and IKK? mRNA by RT-PCR: The results showed that there was a marked rise in the expression of TNF-? and IKK? mRNA in the model group.The expression of TNF-? and IKK? mRNA in each dose group(11.9,23.8,47.6 g/kg)and dexamethasone(5 mg/kg)group decreased significantly.The data showed that LPS can markedly increase the expression of inflammatory genes,while LDP can reduce the expression of these genes in a dose-dependent manner.5.Detection of NF-?B signaling pathway related proteins by Western blot: The results showed that,Pretreatment with LDP and dexamethasone decreased the I?B? phosphorylation ratio(P<0.01).In addition,phosphorylated NF-?B p65 was markedly upregulated in both the nuclear and cytoplasmic protein in the LPS group and down-regulated in the pretreated groups(P<0.01),and the effect of LDP(47.6 g/kg)was equal to that of LDP(23.8 g/kg)group.6.Detection of the expression of NF-?B p65 protein by immunofluorescence: Immunofluorescent confocal microscopy indicated that the expression of p65 in the control group was low and mainly existed in the cytoplasm.The expression of p65 in the LPS group was the highest and mainly existed in the nucleus.The nuclear p65 levels in the pretreatment groups were significantly lower than in the LPS group.The expression of p65 in the LPS group was the highest and mainly existed in the nucleus.The nuclear p65 levels in the LDP groups were significantly downregulated compared with the LPS group.The images indicated that the activated p65 proteins were located in the kidney tubules and cortices.7.Preparation technology of LDP granule: process conditions: raw materials?silicon dioxide?mannitol=1: 0.5: 0.5,using 85% ethanol as a wetting agent.The relative critical humidity was 67.8%.Conclusion: the survival test shows that LDP has a certain protective effect on mice with fatal endotoxemia,and can significantly improve the survival rate.A significant reduction in TNF-?,IL-6 and IL-10 suggested that LDP treatment has anti-inflammatory effect and has a good prognosis in LPS-induced AKI.Histological analysis also revealed that LDP could reduce kidney injury.In addition,LDP can also significantly reduce the expression level of inflammatory genes and NF-?B signaling pathway related proteins,and inhibit the migration of NF-?B protein into the nucleus.These results indicate that LDP prevents LPS-induced AKI by suppressing NF-?B activation.Therefore,LDP can effectively attenuate AKI caused by LPS,and may be a potential therapeutic option in the treatment of LPS induced AKI.The dosage form used in this experiment is granule,the technology is simple and feasible,which could provide a basis for the industrial production of LDP.