The Protective Effects Of Lycium Barbarum Polysaccharides On Hippocampal Neurons And Cognitive Functions In The OVX Mice

BackgroundThe decrease of estrogen in postmenopausal women leads to the decrease of cognitive function.Clinical studies have found that hormone replacement therapy for cognitive impairment is a double-edged sword,and could even increase the incidence of dementia.Many studies have found that some active components in natural food and medicinal materials could protect nerve injury and cognitive function damage in bilateral ovariectomy(OVX)rat model,which provides an important exploration direction for our research.Lycium barbarum is a characteristic industry in Ningxia province.The main active component of it is lycium barbarum polysaccharide(LBP),which has many biological activities,such as anti-inflammation,anti-oxidation,anti-aging and so on.However,its protective effect on cognitive impairment caused by estrogen reduction has not been fully clarified.Objective1.To determine whether LBP can improve the cognitive impairment of OVX mice.2.Based on transcriptomics and proteomics,to analyze the mechanism of LBP's protective effects on hippocampal neurons of OVX mice.Methods1.OVX mouse model establishment and grouping: Female ICR mice were randomly divided into three groups(45mice,12-week-old): SHAM,OVX and OVX+LBP group,with 15 mice in each group.In vitro cultured cells were divided into 7 groups: Con group,AZD group,LBP group,LPS group,AZD9+LPS group,AZD+LBP group,AZD+LBP+LPS group.2.Behavioral test: the novel object recognition test and the open field test were used to test the motor ability,non-spatial learning and memory functions of mice.3.ELISA was used to detect the levels of estradiol in the serum of mice and the levels of TNF-?,IL-1? and IL-6 in mouse astrocytes medium,WST microplate method was used to detect the content of SOD in serum of mice,and TBA colorimetric method was used to detect the content of malondialdehyde(MDA)in serum of mice.4.Three mice in SHAM group and three mice in OVX group were randomly selected.The total RNA of hippocampus was extracted by illumina Novaseq? 6000.The fragments were sequenced by PE150 sequencing mode.5.Three mice in OVX group and OVX+LBP group were randomly selected and the total protein in hippocampus was extracted.TMT quantitative proteome analysis was carried out by high resolution mass spectrometer(Q Exactive Plus).6.H&E,Nissl,TUNEL staining and transmission electron microscope were used to observe the neuron morphology,Nissl body count,apoptotic cell count and neuron ultrastructural changes in the CA1 and CA3 regions of the hippocampus of the three groups of mice.7.The m RNA expression of TLR4/NF-?B signaling pathway proteins and proteomics differential proteins in the hippocampus of the three groups of ICR mice were detected by q PCR.8.The positive expressions of Neu N,SYP,BDNF,ER?,ER?,TLR4,My D88,NF-?B,IL-6,IL-1?,TNF-? and GFAP in hippocampal CA1 and CA3 regions of ICR mice were detected by immunohistochemistry.9.Western blot was used to detect the expression of BDNF,SYP,PSD95,Caspase3,Bax,Bcl-2,TLR4,My D88,NF-?B,Mfn1,Mfn2,Drp1,p-Drp1,Opa1,Beclin1,p62 and Lc3 proteins in hippocampal tissue of ICR mice and HT-22 cells.Results1.In the open field test,the total travel time and central area activity time of the OVX group were less than those of the SHAM group,and the total travel time and central area activity time of the OVX+LBP group were longer than those of the OVX group.In the novel object recognition test,the DI and PI values of mice in the OVX group were lower than those in the SHAM group and the OVX+LBP group.2.The serum levels of E2 in the mice of the OVX group and the OVX+LBP group were lower than that in the SHAM group.Compared with mice in the SHAM group and the OVX+LBP group,the OVX group had lower serum SOD activity and higher MDA serum levels.3.Compared with the SHAM group and the OVX+LBP group,the number of hippocampal neurons and Nissl bodies in the OVX group was significantly reduced,and the number of apoptotic cells was significantly higher in the OVX group.The disappearance of bilateral structure of hippocampal neurons was observed in the OVX group by TEM,and also the breakage,dissolution and formation of focal vacuoles.4.The differential gene KEGG enriched in the hippocampus of the OVX group and the SHAM group.The main differently expressed genes were: NOD-like receptor signaling pathway,Toll-like receptor signaling pathway,tumor necrosis factor signaling pathway,NF-?B signaling pathway.5.The m RNA and protein levels of TLR4/NF-?B signal pathway related factors in hippocampus of OVX group were higher than those in SHAM group and the OVX + LBP group.6.The immunohistochemical results showed that compared with the SHAM group and the OVX+LBP group,the positive expression of Neu N,BDNF and SYP in the hippocampus of the OVX group was significantly lower.In the WB results,the expression of BDNF,SYP and PSD95 proteins in hippocampus of the OVX group were lower than those of the SHAM group and the OVX+LBP group.7.Compared with the mice of OVX group,the proteomics cluster analysis of hippocampal tissue in the OVX+LBP group showed that there were 8 up-regulated and 3 down-regulated proteins.The mitochondrial fusion protein Mfn1 and its related factors were detected.In the hippocampus of the three groups of mice,the expression of Drp1,p-Drp1,and p62 in the OVX group were higher than those in the SHAM and the OVX+LBP group.The protein expressions of Mfn2,Mfn1,Opa1,Lc3 b and Beclin1 in the OVX group were lower than those in the SHAM group and the OVX+LBP group.8.Compared with the AZD group and LPS group,the release of IL-1?,IL-6 and TNF-? in mouse astrocytes in the AZD+LBP group and AZD+LBP+LPS group were reduced.9.In HT-22 cells,compared with the AZD group and LPS group,SYP and PSD95 expression was slightly higher,and the expression of BDNF was increased in the LBP group,AZD+LBP and AZD+LBP+LPS group.Compared with the AZD group and LPS group,the expression of Drp1 and p-Drp1 in the LBP group,AZD+LBP group and AZD+LBP+LPS group was lower.Lc3 b and Beclin1 were lower only in AZD+LPS and AZD+LBP+LPS groups than in those of the Con group.Compared with the Con group,the expression of p62 in AZD group,LPS group and AZD+LPS group was higher.Conclusion1.LBP can improve the cognitive function of OVX mice.2.LBP can protect the hippocampal neurons of the OVX mice through anti-oxidation,anti-inflammation and anti-apoptosis.3.LBP may reduce hippocampal neuron injury in the OVX mice by stabilizing mitotic/fusion imbalance and autophagy.