| ObjectiveThe aim of this study was to investigate the effect of paeoniflorin(PAE)on cognitive dysfunction in MRL/lpr lupus-prone mice.MethodsBy normalizing the NPSLE transcriptomics data in the comprehensive gene expression database and normal samples,the expression data of the obtained genes were incorporated into,the Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform to screen the potential target information of paeoniflorin,and by univariate logistic regression,the paeoniflorin target genes with the ability to significantly differentiate between NPSLE and normal tissues were screened,and the obtained genes were classified according to the hazard ratio.were classified.The immune infiltration analysis of the paeoniflorin target scores was then performed,and the ss GSEA algorithm calculated the immune cell infiltration level of the samples.The pathways associated with the obtained paeoniflorin target scores were enriched to explore the expression of pathways then grouped by different paeoniflorin target scores.MRL/lpr lupus-susceptible mice and C57BL6 mice were purchased and the MRL/lpr lupus-susceptible mice were randomly divided into a paeoniflorin(PAE)group,an MRL/lpr group and a C57BL6 mouse as a normal control(control)group.From the ninth week,the PAE group was gavaged with paeoniflorin(20 mg/kg),and the MRL/lpr group and control group were each gavaged with the same volume of saline.The cognitive impairment of each group was measured by Morris water maze (MWM)and open field text,and the histological lesions in the hippocampus were evaluated by HE staining.The expression of cytokines such as IL-1β,IL-6 and TNF-α in the serum of each group of mice was measured by enzyme-linked immunosorbent assay(ELISA).The expression of PI3 K,AKT,Bcl-2 and BAX in the hippocampus of each group of mice was detected by western blotting.The inflammatory model of HT-22 hippocampal neuronal cells was constructed and grouped into normal group,lps group and lps+paeoniflorin group,and the expression of Il-1β,Il-6 and TNF-α was measured by ELISA in the supernatant inflammatory factors of cell cultures of each group.The expression of PI3 K,AKT,Bcl-2 and BAX in each subgroup of cells was measured by western blotting.ResultsOne-way logistic regression analysis showed that the low OR group included SIRT1,RNF216 and MAPK8 genes,while the high OR group included HTRA3,RHOA,STAT3,FAS,BAX,ATP1A3,ATP1A1,FASLG,MET,ICAM1 and PTGER2,a total of 11 genes,and the high OR group scored mainly in the NPSLE group,while the low OR group score was down-regulated in the NPSLE group.There was an overall positive correlation between the peonidin targeting scores and the infiltration levels of 28 immune cells,with higher levels of immune cell infiltration in the high peonidin targeting score group than in the low score group.Differential analysis showed that 2314 differential genes were obtained in the different paeoniflorin target groups,which were mainly enriched in PI3K-Akt signaling pathway,TNF signaling pathway and NF-kappa B signaling pathway.In animal experiments,the increase in platform finding time and the number of times crossing the platform in the MRL/lpr group of mice in the Morris water maze experiment was(1.4±0.4),and the number of times crossing the central zone in the open field experiment was(15.83±3.429),compared with the mice in the C57/BL6 group,the number of times crossing the platform was reduced and the difference was statistically significant P<0.001,and the score in the open field experiment was decreased and the difference was The number of neurons in the hippocampal tissue of mice in the MRL/lpr group was significantly reduced,and the number of nuclei in the hippocampal tissue of mice in the MRL/lpr group was significantly reduced.The number of neurons in the hippocampal tissue of the MRL/lpr group was significantly reduced,the nuclei of the mice were condensed,the chromatin was condensed,and the number of apoptotic cells was significantly increased,the IL-1 level in the blood supernatant was 108.4±2.249 pg/m L,the IL-6 level was 59.63±2.013 pg/m L,and the TNF-α level was 33.96±5.498 pg/m L,compared with the C57/BL6 group.),increased IL-6 content(P<0.001)and increased TNF-α(P<0.05).The number of neurons in hippocampal tissue was increased,the number of cells was increased and the number of apoptosis was significantly reduced in the paeoniflorin-administered group of mice.The IL-1 content in blood supernatant was 33.91±1.457 pg/m L,IL-6 content was 46.12±3.26 pg/m L and TNF-α content was 15.39±0.0546 pg/m L,compared with the MRL/lpr group of mice,the IL-Western blot results showed that compared with the C57/BL6 group,the expression levels of PI3K(P<0.001),AKT(P<0.001)and BCL-2(P<0.05)were decreased in the MRL/lpr group,while the expression level of BAX was increased.levels were decreased and BAX expression levels were increased,and the differences were statistically significant(P<0.001).Compared to the MRL/lpr group,the PI3K(P<0.001),Akt(P<0.05)and Bcl-2(P<0.05)protein levels were significantly increased and BAX expression levels were decreased in the paeoniflorin administration group(P<0.001).In the cell experiments,the results by flow cytometry technique showed that the apoptosis rate in the LPS-induced group was 59.08±1.827%,which was increased in the LPS-induced group compared with the hippocampal neuronal cells in the CON group(P<0.01),and the apoptosis rate in the LPS+PAE group was 45±3.999%,which was decreased in the LPS+PAE group compared with the LPS group(P< The analysis of ELISA results showed that the supernatant IL-1β content of cell cultures in the LPS group was 6.726±1.715 pg/m L,IL-6 content was 12.95±0.00715 pg/m L and TNF-α content was 14.09±0.1697 pg/m L,compared with the CON group,the supernatant IL-1β content of cell cultures in the LPS group increased(P<0.05).(P < 0.05),IL-6(P < 0.05)and TNF-α(P < 0.05).2.712 ± 0.1727 pg/m L,6.953 ± 1.165 pg/m L of IL-6 and 3.608 ± 0.2186 pg/m L of TNF-α were found in the supernatant of cell culture medium in the LPS+PAE group compared with the CON group,compared with the LPS group,the supernatant IL-1β content(P < 0.05),IL-6 content(P < 0.05)and TNF-α content(P < 0.05)were decreased in the cell culture medium of the LPS+PAE group,and the cell immunofluorescence results showed that the cell PI3 K expression was reduced after LPS induction compared with the cells of the CON group(P < 0.0001),and peonidin administration Western blot results showed that the expression levels of PI3K(P < 0.0001),AKT(P < 0.001)and Bcl-2 protein were decreased(P < 0.0001)and BAX protein expression was increased(P < 0.0001)in cells from the LPS group compared to the CON group.Compared with the LPS group,PI3K(P<0.0001),Akt(P<0.0001)and Bcl-2 protein levels were significantly higher(P<0.001)and BAX expression levels were lower(P<0.001)in the LPS+PAE group.Conclusion1.Paeoniflorin may improve HT-22 neuronal apoptosis by reducing inflammation.2.Paeoniflorin can reduce neuronal inflammation and improve the apoptosis of HT-22 nerve cells. |
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