Zexieyin Formula From Huangdi Neijing Alleviates Cognitive Impairment By Regulating Hippocampal Inflammation And Neurogenesis

ObjectiveCognitive impairment(CI),a common clinical symptom of a variety of diseases,whose incidence increasing year by year,has become a serious threat to human life and health.CI is related to many factors such as cerebral microvascular occlusion,neuroinflammation,and neuronal dysfunction.The Chinese medicine community believes that "orifices confused by phlegm" is the key pathogenesis of cognitive disorders,and Professor Wu Haoxin believes that "accumulation of phlegm converted into heat,heat burning the brain,cerebral cord damage,leading to apraxia of the gods" is the pathogenesis evolution process of "orifices confused by phlegm".Previous research has found that Zexieyin formula(ZXYF),from Hangdi Neijing,can alleviate atherosclerosis(AS)and promote vascular inflammation and microcirculation.Improving vascular microcirculation,especially cerebral microcirculation,is conducive to the release of nutritional factors,promoting neurogenesis,and enhancing memory function.Combined with the consistency of the pathogenesis and treatment of TCM,we speculated that ZXYF has the potential to improve CI.The study was aimed to explore the core neuromolecular mechanism of ZXYF in improving CI and the core drugs that play a major role in the compound.MethodsIn this study,ApoE-/-mice fed with high-fat diet(HFD)were used to establish a combined CI model of AS.Meanwhile,CI models were established using a mouse model of chronic scopolamine(SCOP),lipopolysaccharide(LPS)and APP/PS1 mice.Oil red O staining(ORO)and hematoxylin-eosin staining(HE)were used to detect the area of aortic plaque.Morris water maze(MWM)and Y maze were used to detect the cognitive function of mice.Network pharmacology and mRNA sequencing methods were used to screen potential targets for improving cognitive function.Enzyme-linked immunosorbent assay(ELISA)was used to determine the levels of inflammation-associated cytokines in serum.Western blot and immunofluorescence(IF)were used to detect the expression of targeted proteins in the hippocampus of mice.Pharmacological blockade and hippocampal brain region-specific gene knockdown techniques were used to evaluate the relationship between targets and ZXYF to improve CI.Results1.HFD accelerates the formation of AS in ApoE-/-mice,which is more likely to produce CI thanwild type mice.After 8 weeks of dosing,ZXYF or ATO significantly reduced aortic plaquearea in ORO and HE staining,while significantly improving mouse cognitive ability in MWMand Y maze tests.The results of network pharmacology showed that MAPK was highlycorrelated with CI.HFD resulted in abnormal expression of hippocampal MAPK(pERK,pP38,pJNK),NF-kB,synaptic proteins(PSD95,synapsinl),and β-amyloid(Aβ),which were significantly reversed by ZXYF.2.ZXYF has the effect of improving memory and learning deficiency in two CI mouse models induced by LPS and SCOP and APP/PS1 transgenic AD mouse models.ZXYF significantly increased the expression of CaMKII,CREB and PSD95 proteins in the hippocampus,as well as the expression of BrdU/DCX and Ki67 positive cells in the DG region of the hippocampus in mice,and up-regulated the protein expression levels of AMPA receptor GluA1 and PKA in the hippocampus.Cellular results show that ZXYF activates the expression of CaMKII and GluAl proteins in HT22 hippocampal neuronal cells.Inhibition of the AMPA receptor was able to block the effect of ZXYF in the treatment of CI in MWM and blocked the number of BrdU/DCX-positive cells upregulated by ZXYF in the DG region of the hippocampus.3.The results of ZXYF splitting study showed that Pyrolae herba(PH)could produce a CI improvement effect comparable to ZXYF.LPS was able to induce CI,and treatment with PH significantly improved memory and learning function in MWM test.LPS increased TNF-αand IL-1 β expression in serum and hippocampus,whereas PH reversed the LPS-induced increases in TNF-α and IL-1 β and the activation of glia in the hippocampus.The results of mRNA sequencing and RT-PCR showed that LPS significantly increased the gene expression of TREM2,and PH reversed the up-regulation of TREM2 gene expression by LPS.The protein results showed that LPS up-regulated the expression of TREM2 and its related signaling,which were all reversed by PH.LPS significantly decreased the quantities of BrdU and DCX co-positive cells and synapsin1 protein in hippocampal dentate gyrus(DG),which was significantly reversed by PH.Meanwhile,PH significantly increased the co-positive cells of Ki67 and DCX in the DG region.LPS significantly reduced the learning and memory ability of mice in MWM and Y maze tests,while specific knockdown of hippocampal TREM2 gene expression could improve the learning and memory deficiency induced by LPS.Conclusion1.HFD can promote the formation of CI along with AS production in ApoE-/-mice.It was suggested that"accumulation of phlegm converted into heat,heat burning the brain,cerebral cord damage".ZXYF can alleviate cognitive impairment by regulating MAPK signaling in the hippocampus and reversing synaptic protein and Aβ protein.It meant that ZXYF alleviates cognitive impairment by strengthening spleen to removing dampness.2.ZXYF inhibits hippocampal inflammation and promotes neurogenesis and enhances the transmission of postsynaptic protein factors in the mouse hippocampus by activating and autophosphorylating the postsynaptic associated CaMKII to activate and improve the AMPA receptor(GluA1),further upregulates PKA/CREB signaling,thereby alleviating CI.It meant that ZXYF alleviates cognitive impairment by clearing heat and reducing brain damage.3.PH,reducing the activation of microglia in the hippocampus by inhibiting the TREM2 signaling,and further promoting neurogenesis and proliferation,thereby enhancing the expression of presynaptic proteins by reducing the secretion of pro-inflammatory cytokines and the activity of astrocytes,can produce CI treatment effect similar to ZXYF.It meant that PH plays a critical role in clearing heat to reducing brain damage in ZXYF.