Study On Inflammation Mechanism Of Disorders Caused By The Liver Failing To Convey And Disperse-Research On Paeonol Treating Depression With Liver-qi Stagnation Through TLR4-NF-κB Signaling Pathway

Objuctive: To study the effect of paeonol on TLR4-NF-κ B signaling pathway in the lipopolysaccharide-induced depression mouse model and primary cell model,and to verify the hypothesis of disorders caused by the liver failing to convey and disperse inflammation mechanism.Methods: 1.Use minocycline(50mg/kg),fluoxetine(20mg/kg),low dose of paeonol(10mg/kg),high dose of paeonol(20mg/kg)to pre-intraperitoneal injection for mice(injected with 0.9% physiological saline 10 mg/kg in the control group and model group),and then establish a depression liver-qi stagnation model by intraperitoneal injection of lipopolysaccharide(0.5 mg/kg).The forced swimming test and suspension test were used to evaluate the model.2.Using ELISA kit to detect serum TNF-α,IL-1β,IL-6,IL-10 levels in the mouse model of depression;Western Blot was used to determine the protein expression levels of p65、 Iκ Bα 、 IKKβ 、NLRP3 in the hippocampus of mice;real-time quantitative PCR was used to detect the mRNA expression of p65、Iκ Bα 、IKKβ 、NLRP3 in the hippocampus of mice.3.Primary neurons in the hippocampus cultured in the vitro,after cells maturation,given low-dose(0.5μ M)paeonol,high-dose(5μ M)minocycline(10μ M),fluoxetine(5μ M)in 24 hours advance.Cells were then treated with LPS(10μ g/ml)and were incubated for 24 hours to establish a depressive liver-qi stagnation cell model.Cell viability was measured using MTT.4.The ELISA kit was used to detect the content of TNF-α,IL-1β,IL-6 and IL-10 in the cell culture supernatant;Western Blot detected the protein expression levels of p65、Iκ Bα 、IKKβ 、NLRP3 in the cell model;real-time quantitative PCR detection of p65、 Iκ Bα 、 IKKβ 、 NLRP3 mRNA expression levels in the cell model.Results: 1.Depression mouse model evaluation: compared with the control group,the immobility time of the LPS model group significantly longer,the difference was statistically significant(p <0.01),minocycline group,fluoxetine group,Compared with the LPS model group,the paeonol low-dose group,high-dose paeonol group had significantly decreased immobility time(P<0.05).2.Detection results of ELISA kit: compared with the control group,LPS group,minocycline group,fluoxetine group,paeonol low-dose group,and paeonol high-dose group mice serum TNF-The content of α,IL-1 β,IL-6 and IL-10 increased significantly(P<0.01).Compared with LPS model group,serum TNF-α,IL-1β,and IL-6 levels were significantly lower in minocycline group,fluoxetine group,paeonol low-dose group,and high-dose paeonol group.Decreased(P<0.05),while IL-10 content increased significantly(p<0.05).Western Blot detection results: compared with the control group,the protein expression levels of p65,Iκ Bα,IKKβ,and NLRP3 in the LPS model group were significantly increased(P<0.05),compared with the LPS model group,the fluoxetine group,The protein expressions of p65,Iκ Bα,IKKβ and NLRP3 in the minocycline group,low-dose paeonol group,and high-dose paeonol group were significantly decreased(P<0.05),while the low-dose paeonol group was lower than the low-dose group.In the minocycline group,fluoxetine group and paeonol high-dose group,the protein expression levels of p65,Iκ Bα,IKKβ and NLRP3 were higher,the difference was statistically significant(P<0.05).Real-time quantitative PCR results: Compared with the control group,the mRNA expression levels of p65,Iκ Bα,IKKβ and NLRP3 in the other six groups increased significantly(P<0.05),compared with the LPS model group,the fluoxetine group.The mRNA expression levels of p65,Iκ Bα,IKKβ and NLRP3 were significantly lower(P<0.05)in the Minocycline group,the low-dose paeonol group,and the high-dose paeonol group,while the low-dose paeonol group Compared with the minocycline group,fluoxetine group,and paeonol high-dose group,the mRNA expression levels of p65,I κ B α,IKK β and NLRP3 were higher,and the difference was statistically significant(P<0.05).3.MTT assay of primary hippocampal neurons in vitro: compared with control group,LPS model group,minocycline group,fluoxetine group,paeonol low dose group and paeonol high dose group The survival rate was significantly lower(P<0.05).The survival rate of fluoxetine group,minocycline group,paeonol low-dose group and high-dose paeonol group was significantly higher than that of LPS model group(P<0.05).The cell viability of the fluoxetine group and the high-dose paeonol group was significantly higher than that of the low-dose paeonol group(P<0.05).4.Detection results of cell model by ELISA kit: The content of TNF-α,IL-1β,IL-6,and IL-10 in the supernatant of the cells was compared with the control group,LPS model group,minocycline group,and fluorine The levels of TNF-α,IL-1β,IL-6 and IL-10 in the cell supernatants of the cetatin group,the low-dose paeonol group and the high-dose paeonol group were significantly increased(P<0.01).The fluoxetine group,minocycline group,paeonol low-dose group,and paeonol high-dose group had significantly lower levels of TNF-α,IL-1β,and IL-6 compared with LPS model group,while IL-10 significant increase(P <0.05).The levels of TNF-α and IL-6 in the supernatant of paeonol low-dose group were significantly higher than those of minocycline group,fluoxetine group and paeonol high-dose group,while the content of IL-10 was significantly lower than these three groups.Group(P < 0.05).Western Blot test on cell model: compared with the control group,the protein expression levels of p65,Iκ Bα,IKKβ and NLRP3 in the LPS model group were significantly increased(P<0.05),compared with the LPS model group,The protein expressions of p65,Iκ Bα,IKKβ and NLRP3 in the control group,Minocycline group,Paeonol low-dose group,and Paeonol high-dose group were all significantly decreased(P<0.05),while the paeonol low-dose group The protein expressions of p65,Iκ B α,IKK β,NLRP3 were significantly higher than those of minocycline group,fluoxetine group and paeonol high dose group(P<0.05).Results of real-time quantitative PCR on cell model: Compared with the control group,the mRNA expression levels of p65,I κ B α,IKK β,NLRP3 in the other five groups increased significantly(P<0.05),compared with the LPS model group.The mRNA expressions of p65,Iκ Bα,IKKβ,NLRP3 in the fluoxetine group,minocycline group,paeonol low-dose group and high-dose paeonol group were significantly lower(P<0.05),while paeonol was low-dose Compared with the minocycline group,the fluoxetine group,and the paeonol high-dose group,the mRNA expression levels of p65,Iκ Bα,IKKβ,NLRP3 were higher,and the difference was statistically significant(P<0.05).Conclusions: Paeonol has a positive therapeutic on depression liver-qi stagnation,which is consistent with previous studies.The therapeutic effect of paeonol on depression may be dose-dependent.Paeonol inhibits the expression of p65,Iκ Bα,IKKβ,NLRP3 by inhibiting the activation of TLR4 and NF-κ B signaling pathways,thereby inhibiting the production of pro-inflammatory cytokines and promoting the production of anti-inflammatory cytokines to relieve the depressive symptoms.