| Objective: Clinical patients may have language and visual memory,visual space abstraction,psychomotor function and attention thought process disorders,that is,postoperative cognitive dysfunction(Postoperative cognitive dysfunction,POCD).As one of the most common postoperative complications,POCD seriously affects the quality of life of patients after operation.Therefore,it is an important clinical topic to explore the pathogenesis,prevention and treatment of POCD.The main purpose of this study is to explore the Mongolian medicine Erdun-Wril preparation and its basic prescription to improve the occurrence of postoperative cognitive dysfunction by inhibiting the inflammatory response of TLR4/NF-κB signal pathway,in order to provide some basic experimental data for clinical prevention and treatment of POCD.Methods: 60 male C57BL/6 mice of SPF grade 6～8weeks,weighing 1～21 g,were randomly divided into 5 groups: normal control group(group C),lateral ventricular injection of lipopolysaccharide(LPS)+ left nephrectomy group(group M),Mongolian medicine Erdun-Wril preparation group(group Z),Mongolian medicine Erdun-Wril basic prescription group(group J),and dexmetomidine positive control group(group D).All mice were fed in clean environment,the light and dark time were alternate for12 hours,the water was consumed freely,and the temperature of the feeding room was20～24 ℃.The experiment was carried out after 7 days of adaptive feeding.The mice were trained to navigate by water maze 5 days before operation.The mice in group Z and group J were continuously intragastric with equal doses of Erdun-Wril preparation and Erdun-Wril basic prescription(8.6 mg/10g)starting from 6 days before surgery,respectively,and the mice in group C and group M were intragastric with equal amounts of distilled water until the day of operation.Except for group C,mice in all groups were injected with LPS 2 μg(1μg/μL)into the lateral ventricle 1 hour before operation,followed by left nephrectomy under chloral hydrate anesthesia.Mice in group D were injected intraperitoneally with 25 μg/kg dexmetomidine after lateral ventricle injection of LPS before operation.The postoperative behavior test was performed on the 1st day,2nd and 3rd day after operation.The escape latency,swimming speed,the number of times crossing the platform and the percentage of staying time in the target quadrant were recorded,and the cognitive function and motor ability of each group were evaluated.After 6 hours and 3 days after operation,blood samples were taken from fundus vein under anesthesia and mice were killed to take hippocampal tissue or brain.HE staining was used to detect the degeneration and necrosis of nerve cells and the infiltration of inflammatory cells in the hippocampus of mice.The changes of inflammatory cytokines IL-1β,IL-6 and TNF-α in serum of mice were detected by Elisa.The expression of TLR4,NF-κBp65 and MyD88 related to TLR4/NF-κB signal pathway was detected by Western Blot.RT-PCR method was used to detect the expression of TLR4/NF-κB signal pathway related proteins TLR4,NF-κBp65 and MyD88 at m RNA level.Results: 1.Results of water maze: the positioning navigation training was carried out 5 days before the operation,and the latency of the mice to find the platform gradually shortened with the extension of the training days.Compared with the first day,the escape latency of the mice in each group on the fifth day of training was shortened,and the difference was statistically significant(P<0.05).There was no statistically significant difference in the escape latency of the mice in each group on the fifth day of training(P>0.05).The behavioral results of positioning navigation and space exploration on the 1st,2nd and 3rd day after operation showed that compared with group C,the escape latency of group M mice was prolonged,the number of times of crossing the platform was reduced,and the percentage of residence time in the target quadrant(the first quadrant)was decreased,the difference was statistically significant(P<0.05);Compared with group M,the escape latency of mice in group Z,J and D was shortened,the number of times of crossing the platform was increased,and the percentage of target quadrant residence time was increased,the difference was statistically significant(P<0.05);There was no statistically significant difference in behavioral indicators such as escape latency,number of times of crossing the platform and percentage of target quadrant residence time between group Z,group J and group D(P>0.05).There was no significant difference in swimming speed among the groups on the 1st,2nd and 3rd day after operation(P>0.05).2.Elisa result: the levels of IL-1β,IL-6 and TNF-α inflammatory cytokines in M group were significantly higher than those in C group at 6 h and 3 d after operation(P<0.05).Compared with group M,the levels of IL-1β,IL-6 and TNF-α in group Z,group J and group D were significantly lower at 6h and 3d after operation(P<0.05).Compared with group J,the levels of inflammatory cytokines 6h IL-1β,IL-6 and TNF-α in group Z had no significant difference after operation(P>0.05),but the levels of IL-1β,IL-6 and TNF-α in group Z were significantly lower than those in group J on the 3rd day after operation(P<0.05).3.Western Blot results: compared with group C,the protein levels of TLR4,MyD88 and NF-κBp65 in group M were significantly higher at 6h and 3d after operation(P<0.05).Compared with M group,the protein levels of 6h TLR4,MyD88 and NF-κBp65 in Z group,J group and D group decreased significantly after operation(P<0.05).Compared with group M,the protein levels of TLR4,MyD88 and NF-κBp65 in group Z decreased significantly on the 3rd day after operation(P<0.05).Compared with group M,the level of TLR4 protein in group D and group J decreased at the 3rd day after operation,and the difference was not statistically significant(P>0.05).The level of MyD88 and NF-κBp65 protein decreased significantly(P<0.05).Compared with group J,the protein levels of 6h TLR4,MyD88 and NF-κBp65 in group Z decreased significantly after operation(P<0.05).Compared with group J,the level of TLR4 protein in group Z decreased at the 3rd day after operation,and the difference was not statistically significant(P>0.05).The level of MyD88 and NF-κBp65 protein decreased significantly(P<0.05).4.RT-PCR results: Compared with group C,the m RNA level of TLR4,MyD88 and NF-κBp65 protein increased significantly in group M at 6h and 3d after operation(P<0.05).Compared with group M,the m RNA level of TLR4,MyD88 and NF-κBp65 protein decreased significantly in group Z,J and D mice at 6h and 3d after operation(P<0.05).Compared with group J,the m RNA level of TLR4,MyD88 and NF-κBp65 protein decreased without statistical significance in group Z at 6h after operation(P>0.05).Compared with group J,the m RNA level of TLR4 protein in group Z decreased at the 3rd day after operation,and the difference was not statistically significant(P>0.05),the m RNA level of MyD88 and NF-κBp65 protein decreased significantly(P<0.05).Conclusion: Mongolian medicine Erdun-Wril preparation and its basic prescription can improve the occurrence of postoperative cognitive dysfunction in mice.TLR4/NF-κB signal pathway is one of the important mechanisms of Mongolian medicine Erdun-Wril in the prevention and treatment of postoperative cognitive dysfunction. |
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