| Alzheimer’s disease（AD）is a neurodegenerative disease related to age,and the initial event of its onset has become the focus of attention.Aβ induced microglial activation leads to activation of intracellular NLRP3 inflammasome,and the production of Caspase-1 and IL-18,IL-1β inflammatory factors into the vicious cycle of inflammatory cascade,which can lead to synaptic and neuronal damage and dysfunction and initiate the course of AD.Neuroinflammation of microglia induced by abnormal deposition of Aβ is an important link in triggering and driving AD pathology.A large number of experimental studies have shown that berberine,baicalin and other effective ingredients of Banxia Xiexin Decoction can play an anti-neuroin fl ammat i on effect.Banxia Xiexin Decoction is a classic prescription characterized by the combination of bitter and bitter health,cold and temperature,and tonifying and reducing heat.It can treat the pathogenesis of AD,such as deficiency and deficiency,deficiency of temper,and accumulation of phlegm and heat,and play the role of invigorating spleen,tonifying deficiency,reducing phlegm and clearing heat.In our previous studies,Banxia Xiexin Decoction can reduce Aβ deposition in hippocampus of APP/PS1 mice,increase the autophagy function of neurons,alleviate synaptic damage,improve cerebral glucose metabolism and relieve cognitive dysfunction,and whether the triggering mechanism is related to Banxia Xiexin Decoction improving Aβ induced microglia-mediated AD neuroinflammation deserves further investigation.ObjectiveThis project intends to observe the effects of Banxia Xiexin decoction on Aβ42 deposition,microglia activation,NLRP3 inflammatory body activation and NFκB/NLRP3-Caspase-1 signaling pathway related proteins in the brain of AD animal model APP/PS1 double transgenic mice.At the same time,Aβ42 was used to induce the activation of BV2 microglia to simulate the in vitro neuroinflammation model of AD,and BAY and MCC950 inhibitors were used as positive drugs for mechanism to further verify and explore the possibility of Banxia Xiexin decoction inhibiting NFκB/NLRP3-Caspase-1 signaling pathway and inflammatory microenvironment.Thus,this provides a reliable molecular biological theoretical basis for treating Banxia Xiexin Decoction from spleen to improve neuroinflammation and cognitive function of AD.Methods1 Grouping to drugs:Ninety 3-month-old APP/PS1 double transgenic mice were randomly divided into model group,Donepezil group,Banxia Xiexin decoction high-dose,medium-dose and low-dose groups,with 18 mice in each group.At the same time,18 wild-type C57BL/6J mice of the same background and age were treated as normal group.Normal group and model group were given 0.5%CMC,donepezil group was given donepezil（0.92 mg·kg-1d·-1）by intragastric administration according to body weight.Banxia Xiexin decoction group was given large dose(12 g·kg-1·d-1),medium dose(6 g·kg-1·d-1)and small dose(3 g·kg-1·d-1)by intragastric administration,once every morning for 3 months.2 Behavioral experiments:Morris water maze experiment were used to study the effects of drug administration on spatial learning and memory ability of APP/PS1 double transgenic mice after 3 months of intervention.3 Aβ42 deposition and microglial activation detection:Immunohistochemistry and Western blot were used to study the effects of drug administration on Aβ42 deposition and expression of microglia activation marker Iba-1 protein in hippocampus of APP/PS1 double transgenic mice after 3 months of intervention.4 Hippocampal NFκB/NLRP3-caspase-1 signaling pathway detection:The effects of drug administration on the expression of NFκB/NLRP3-Caspase-1 signaling pathway related proteins and mRNA in the hippocampus of APP/PS1 double transgenic mice after 3 months of intervention were studied by immunofluorescence double label,Western blot,RT-qPCR and immunoprecipitation.5 Preparation of drug-containing serum:Forty male Sprague-dawley rats（200 ± 20g）were randomly divided into normal group and Banxia Xiexin decoction group.Banxia Xiexin decoction group was given intragastric administration at twice the equivalent dose of adult（70 kg）,and normal group was given 0.5%CMC once a day for 7 days.6 Nontarget metabolomics of Traditional Chinese medicine:The active components in Banxia Xiexin decoction containing serum and containing cerebrospinal fluid were detected by the liquid chromatography-non-targeted metabolomics method.7 Establishment of AD in vitro neuroinflammation model:Aβ42 was used to induce BV2 microglia activation to establish an AD neuroinflammation model in vitro,and CCK-8,Western blot and RT-qPCR were used to evaluate the model.8 Determination of effective dose of drug-containing serum:The activity of BV2 microglia was detected by CCK-8 method,and the safe and effective dose and action time of Banxia Xiexin decoction containing serum were determined.9 Detection of NFκB/NLRP3-caspase-1 signaling pathway in BV2 microglia:Immunofluorescence,Western blot and RT-qPCR were used to study the effects of Banxia Xiexin decoction containing serum on NFκB/NLRP3-Caspase-1 inflammatory signaling pathway related proteins,downstream pro-inflammatory factor proteins and mRNA expression in BV2 microglia induced by Aβ42.10 Mechanism positive drug control related detection:The NFκB inhibitor BAY and NLRP3 inflammatory body inhibitor MCC950,Western blot and RT-qPCR were used to further verify that the Banxia Xiexin decoction containing serum reduced the expression of pro-inflammatory factors through NFκB/NLRP3-Caspase-1 signaling pathway to improve the neuroinflammatory response.Results1 Banxia Xiexin decoction can improve the spatial learning and memory ability of APP/PS1 double transgenic miceMorris water maze results showed that the escape latency and swimming distance of mice in the model group were significantly longer than those in the normal group during the 1st to 5th day of the positioning navigation experiment（P<0.01）.Compared with model group,the escape latency of donepezil group was significantly shortened from day 3（P<0.01）,and the swimming distance of donepezil group was significantly shortened from day 2（P<0.01）.At the same time,the escape latency of mice in high-dose Banxia Xiexin decoction group was shortened on day 4 and 5 compared with that in model group,and the difference was statistically significant（P<0.01）.The swimming distance of mice in high-dose and medium-dose Banxia Xiexin decoction group was significantly shortened on day 3 compared with that in model group（P<0.01）.Morris water maze results showed that compared with the normal group,the number of times of crossing the platform in the model group was significantly reduced（P<0.01）,the percentage of target quadrant residence time in total time decreased significantly（P<0.01）,the swimming distance in target quadrant decreased significantly（P<0.01）.Compared with model group,donepezil group and Banxia Xiexin decoction high-dose group had higher number of crossing platform,percentage of stay time in target quadrant and swimming distance in target quadrant,and the difference was statistically significant（P<0.01,P<0.05）.2 Banxia Xiexin decoction can reduce the deposition of amyloid Aβ42 and activation of microglia in hippocampus of APP/PS1 double transgenic miceImmunohistochemical staining showed that the area of Aβ42 plaque in hippocampus of model group was significantly larger than that of normal group（P<0.01）,the number of Iba-1 positive cells increased significantly（P<0.01）;Compared with model group,the area of Aβ42 plaque in hippocampus of donepezil group and Banxia Xiexin decoction groups was significantly decreased（P<0.01）,the number of Iba-1 positive cells in the hippocampus of donepezil group and Banxia Xiexin decoction large and medium dose groups was significantly decreased（P<0.01）.Western blot showed that the expression of Aβ42 and Iba-1 protein in hippocampus of model group was significantly higher than that of normal group（P<0.01）;Compared with model group,the expression of Aβ42 and Iba-1 proteins in the hippocampus of donepezil group and banxia Xiexin decoction groups were decreased to different degrees,and the difference was statistically significant（P<0.01,P<0.05）.3 Banxia Xiexin decoction down-regulated expression of NFκB/NLRP3-Caspase-1 inflammatory pathway-related proteins and mRNA transcription levels in the hippocampus of APP/PS1 double transgenic miceWestern blot showed that the protein expression of IL-1β,IL-18 and p-NFκB,NLRP3,ASC,Caspase-1 was significantly increased compared with normal group（P<0.01,P<0.05）.Compared with model group,the protein expressions of IL-1β,IL-18 and p-NFκB,NLRP3,ASC,Caspase-1 in the hippocampus of donepezil group and Banxia Xiexin decoction groups were significantly decreased,and the differences were statistically significant（P<0.01）.RT-qPCR results showed that the transcription levels of IL-1β,IL-18 and NLRP3,ASC,Caspase-1 mRNA in the hippocampus of model group were higher than those in the normal group（P<0.01,P<0.05）.Compared with model group,the transcription levels of NLRP3 and ASC mRNA in hippocampus of donepezil group were significantly decreased（P<0.01）,the transcription level of IL-1β,IL-18 and ASC mRNA in the hippocampus of mice in Banxia Xiexin decoction high-dose and medium-dose groups was decreased（P<0.01,P<0.05）.4 Banxia Xiexin decoction can reduce the interaction between NLRP3 inflammatory bodies in the hippocampus of APP/PS1 double transgenic miceCo-IP results showed that when ASC protein was precipitated,NLRP3 and Procaspase-1 proteins interacting with ASC were significantly increased in the model group compared with the normal group,and the difference was statistically significant（P<0.01）.Compared with model group,the NLRP3 and Procaspase-1 proteins interacting with ASC in hippocampus of donepezil group and Banxia Xiexin decoction groups were decreased to different degrees,with statistically significant differences（P<0.05,P<0.01）.5 LC/MS was used to detect the active components in the Banxia Xiexin decoction containing serum and cerebrospinal fluidThe results of LC/MS experiment indicated that the active components in Banxia Xiexin decoction containing serum and cerebrospinal fluid mainly included flavonoids,nucleosides and alkaloids,such as baicalin,rutin,berberine,allantoin,thymine,uracil,inosine,ferulic acid and succinic acid.etc.6 The optimal concentration and time of Aβ42 induced activation of BV2 microglia to establish a model of AD neuroinflammation in vitroBV2 microglia were induced by Aβ42 to induce neuroinflammatory response.The method of 5μM Aβ42 to induce BV2 microglia for 24 h was used to establish the neuroinflammatory model of AD in vitro.CCK-8 showed that the cells were incubated with 1.25-5 μM Aβ42 for 16 h or 24 h,compared with the normal group,the cell OD value of 10 μM and 20 μM was lower than that of the normal group,and the difference was statistically significant（P<0.05,P<0.01）.Western blot results showed that when the concentration of Aβ42 was 5 μM,the expression of Iba-1,IL-1β and IL-18 protein was significantly higher than that of the normal group（P<0.01）,and also had statistical significance compared with 1 μM and 10 μM（P<0.01）.Meanwhile,RT-qPCR results showed that the mRNA levels of IL-1β and IL-18 in BV2 cells after 5 μM AP42 treatment for 24 h were higher than those in the normal group,and the differences were statistically significant（P<0.01,P<0.05）.Therefore,combined with the results of CCK-8 experiment,BV2 microglia were induced by 5 μM Aβ342 for 24 h under the condition that BV2 cells were not damaged but the expression of inflammatory factors was increased,and the in vitro neuroinflammation model like AD was established.7 The optimal concentration and time of Banxia Xiexin decoction containing serum on BV2 microgliaCCK-8 results indicated that compared with the normal serum,Banxia Xiexin decoction containing serum of 2.5%,5%,10%,15%and 20%had no toxic effects on normal BV2 cells for 16 and 24h.After 48 h treatment,compared with normal serum,20%Banxia Xiexin decoction could reduce the cell activity,and the difference was statistically significant（P<0.01）.According to the above results,2.5%～15%Banxia Xiexin decoction containing serum did not produce toxicity to BV2 cells when treated with BV2 cells for 16,24 and 48 hours.After comprehensive consideration,10%Banxia Xiexin decoction containing serum was treated with BV2 cells for 24 hours as the subsequent experimental condition.8 Banxia Xiexin decoction containing serum can down-regulate the expression of NFκB/NLRP3-Caspase-1 inflammatory pathway associated protein and mRNA in Aβ42-induced BV2 microgliaImmunofluorescence and Western blot results showed that the expression of IL-1β,IL-18 and p-NFκB,NLRP3,ASC,Caspase-1 protein in the model group was significantly higher than that in the normal group（P<0.01）.Compared with the model group,the expression of IL-1β,IL-18 and p-NFκB,NLRP3,ASC,Caspase-1 protein in Banxia Xiexin decoction containing serum was decreased to different degrees（P<0.01,P<0.05）.RT-qPCR results showed that the transcription levels of IL-1β,IL-18 and NLRP3,ASC,Caspase-1 mRNA in the model group were higher than those in the normal group（P<0.05）,however,the Banxia Xiexin decoction containing serum reduced the transcription level of IL-1β,IL-18 and NLRP3,Caspase-1 mRNA,and the difference was statistically significant（P<0.01,P<0.05）.9 The effects of banxia Xiexin Decoction serum on NFκB/NLRp3-caspase-1 inflammatory pathway related proteins and mRNA expression in BV2 cells induced by Aβ42 were observed after the application of mechanopositive drugsAfter treatment with NFκB inhibitor BAY and NLRP3 inflammasosome inhibitor MCC950,Western blot results showed that the protein expression of p-NFκB,NLRP3,ASC and Caspase-1 in the model group was significantly higher than that in the normal group（P<0.01）,the expression of IL-1β and IL-18 also increased in different degrees（P<0.01,P<0.05）.Compared with model group,Banxia Xiexin decoction containing serum and BAY significantly reduced the expression of p-NFκB,NLRP3,ASC and Caspase-1 protein（P<0.01）,MCC950 decreased the expression of ASC and Caspase-1 protein（P<0.01）,the expression of downstream pro-inflammatory factor IL-1β was also decreased in all treatment groups（P<0.01,P<0.05）,BAY also decreased the expression of IL-18 protein（P<0.01）.RT-qPCR results showed that the transcription levels of NLRP3 and ASC mRNA in model group were significantly higher than that in normal group（P<0.01）,the transcription levels of IL-1β and IL-18 mRNA were also significantly increased（P<0.01）compared with the model group,the transcription levels of IL-1β,IL-18,NLRP3,and ASC mRNA in Banxia Xiexin decoction containing serum and BAY were decreased to different degrees（P<0.01,P<0.05）,and MCC950 can reduce the transcription levels of ASC and IL-1β mRNA.ConclusionsBanxia Xiexin decoction has the effect of improving neuroinflammation to play a neuroprotective role,the mechanism of which may be through reducing the neuroinflammation response of BV2 microglia induced by Aβ42,decreasing the expression of NFκB/NLRP3-Caspase-1 pathway related proteins and its downstream pro-inflammatory factors IL-1β and IL-18,and reducing the interaction of NLRP3 inflammasome. |
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