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Objective To explore the impact of the sialic acid binding lectin-E(Siglec-E)on the inhibitory properties of parthenolide(PTL)against lipopolysaccharide(LPS)-induced M1 polarization of microglia(BV2).Methods ①Single cell sequencing data of Siglece related mouse brain tissue was obtained from Gene Expression Omnibus(GEO)database and divided into the WT group(n=3)and the Siglece-/-group(n=4).The microglia cells were screened,and the enrichment analysis was performed to analyze related differential genes and pathways.BV2 cells were constructed by the shRNA interference technique and were divided into NC-shRNA and Siglece-shRNA to detect the expression level of Siglec-E(Siglece).② NC-shRNA and Siglece-shRNA cells were respectively divided into the Control group,LPS group,PTL group and PTL+LPS group(n=3).The mRNA levels of markers of M1 polarization in microglia,iNOS,IL-1 β and IL-6,were detected by RT-qPCR.Siglecefl/fl and Cx3cr1cre mice were mated to obtain microglia-specific Siglece deletion(Siglecefl/fl×Cx3cr1cre)mice,and LPS-induced neuroinflammation model was established.③ Nine WT and Siglecefl/fl×Cx3cr1cre male mice were assigned to the Control group,LPS group and PTL+LPS group(n=3).RT-qPCR,immunofluorescence assay and Western blotting were used to verify the knock-out effect and polarization-related pathways,and to investigate the mechanism of Siglec-E affecting PTL inhibition of M1 polarization of microglia.Results Compared with the NC-shRNA group,the expression of Siglec-E in the Siglece-shRNA group was significantly decreased(P<0.01),indicating that the Siglec-E knock-down cell model was successfully established.With the stimulation of LPS,mRNA levels ofiNOS,IL-1 β and IL-6 were significantly up-regulated compared with the Control group both in shRNA cells and Siglece-shRNA cells(P<0.01).With the influence of PTL and LPS,the markers of M1 polarization in NC-shRNA cells mentioned before were significantly decreased(P<0.05),while for Siglice-shRNA cells,there were no significant changes in the markers of M1 polarization.PTL inhibited the phosphorylation of JNK and IκB protein(P<0.01)and the nuclear translocation of NF-κB in BV2 cells,down-regulated Siglec-E,and weakened the inhibitory effect.Compared with mice in the WT group,the expression of Siglec-E in microglia of Siglecefl/fl×Cx3cr1cre mice was decreased significantly(P<0.01),and the inhibitory effect of PTL on the phosphorylation of NF-κB in microglia of Siglecefl/fl×Cx3cr1cre mice was also decreased.Conclusion The absence of Siglec-E in microglia attenuates the inhibition of M1 polarization by the MAPK/NF-κB pathway targeted by PTL.
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Background: Regeneration of injuries occurring in the central nervous system is extremely difficult. Studies have shown that the developing cerebellum can be repopulated by a group of Nestin-expressing progenitors (NEPs) after irradiation injury, suggesting that modulating the mobilization of NEPs is beneficial to promoting nerve regeneration. To date, however, effect of exogenous pharmaceutical agonist on NEPs mobilization remains unknown. Parthenolide (PTL), a sesquiterpene lactone isolated from shoots of feverfew. Although it has been shown to possess several pharmacological activities and is considered to have potential therapeutic effects on the regeneration of peripheral nerve injury, its efficacy in promoting central nervous system (CNS) regeneration is unclear. In this study, we aimed to elucidate the role and possible mechanism of PTL on regeneration in injured CNS after irradiation using a developing cerebellum model. Methods: We investigated the radioprotective effects of PTL on the developing cerebellum by immunoblotting as well as immunofluorescence staining and ROS detection in vivo and in vitro experiments, and then determined the effects of PTL on NEPs in Nestin CFP and Nestin GFP fluorescent mice. Inducible lineage tracing analysis was used in Nestin-CreERT2×ROSA26-LSL YFP mice to label and track the fate of NEPs in the cerebellum after irradiation. Combined with cell biology and molecular biology techniques to determine changes in various cellular components in the cerebellum and possible mechanisms of PTL on NEPs mobilization in the injured developing cerebellum. Results: We found that PTL could attenuate radiation-induced acute injury of granule neuron progenitors (GNPs) in irradiated cerebellar external granule layer (EGL) by alleviating apoptosis through regulation of the cells' redox state. Moreover, PTL increased cerebellar Shh production and secretion by inhibiting the PI3K/AKT pathway, thus promoting expansion of NEPs, which is the compensatory replenishment of granule neurons after radiation damage. Conclusion: Collectively, our results indicate that activation and expansion of NEPs are critical for regeneration of the injured cerebellum, and that PTL is a promising drug candidate to influence this process.
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Objective To investigate the effect of cluster needling at scalp points plus gavage of modified Buyang Huanwu decoction on NSC differentiation in rats with ischemic stroke of qi deficiency and blood stasis type.Methods A model of qi deficiency and blood stasis syndrome was made using healthy rats. Dil dye was given for pre-labelling after the success of model making. The rats were randomized into groups A, B, C, D, E and F. Group F consisted of 4 rats and each of the other groups, of 12 rats. A rat model of ischemic stroke of qi deficiency and blood stasis type was made by MCAO at 48 hrs after pre-labelling. After the success of model making, group A was the model group, group B was treated by cluster needling at scalp points, group C was treated by an oral gavage of Buyang Huanwu decoction, group D was treated by an oral gavage of modified Buyang Huanwu decoction, group E was treated by cluster needling at scalp points plus an oral gavage of modified Buyang Huanwu decoction and group F was the sham operation group. The rats were sacrificed at various time points respectively and the materials were taken. By nerve function assessment, double immunofluorescence staining and laser scanning confocal microscopy, a comparative study was conducted to investigate the effects of different treatments on NSC differentiation.Results There was a statistically significant difference in the nerve function score between group B, C, D, E or F and group A after one week of treatment (P<0.01), between group B or E and group A after two weeks of treatment (P<0.05,P<0.01) and between group E and group C or D after one and two weeks of treatment (P<0.01,P<0.05). After one and two weeks of treatment, there were statistically significant differences in Brdu﹢/NeuN﹢ cell count, Brdu﹢/GFAP﹢ cell count, Dil﹢/Brdu﹢NeuN﹢ cell count and Dil﹢/Brdu﹢/GFAP﹢ cell count between group B, C, D or E and group A (P<0.01), between group D or E and group B or C (P<0.01) and between groups E and D (P<0.01).Conclusions Various treatments have a promoting effect on nerve stem cell differentiation in the rats. Of various treatments, cluster needling at scalp points plus gavage of modified Buyang Huanwu decoction has the best therapeutic effect.
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OBJECTIVE@#To investigate the effect of inhibiting high mobility group box-1 (HMGB1) gene expression on the invasion and migration of endometrial carcinoma HEC-1A cells by small hairpin RNA.@*METHODS@#Three specific recombinant plasmids of HMGB1 (pshRNA-1 /HMGB1, pshRNA-2 / HMGB1, and pshRNA-3/HMGB1) were transfected into the endometrial cancer cell lines HEC- 1A by lipofectamine (TM) 2000. The expression of HMGB1 mRNA and protein was decteted by RTPCR and Western blot. The invasion and migration abilities of transfected HEC-1A cells were evaluated using Transwell assay and wound healing assay.@*RESULTS@#RT-PCR and Western blot revealed that the expression of HMGB1 at both mRNA and protein levels was significantly inhibited by HMGB1-pshRNA targeting sequence 1, 2, and 3 (P<0.05), and the levels of 3 mRNAs in the transfection group were 0.192±0.006, 0.055±0.002, and 0.123±0.086, respectively, which were significantly lower than those in the Lipo group (0.268±0.008) and the HMGB1/p-NC group (0.270±0.004). The maximum inhibiton rates of the 3 mRNAs were 28.4%, 79.5%, and 54.1%. The levels of 3 HMGB1 proteins in the transfection group were 0.259±0.129, 0.032±0.002, and 0.104±0.007, significantly lower than those in the Lipo group (0.347±0.007) and the HMGB1/p-NC group (0.349±0.007), and the maximum inhibitory rates were 25.4%, 90.8%, and 70.0%. The transwell chamber assay and wound healing assay showed that the invasion and migration of HEC-1A cells were effectively suppressed by inhibiting HMGB1 expression (P<0.05).@*CONCLUSION@#pshRNA-HMGB1 can effectively inhibit HMGB1 expression at both mRNA and protein levels, and decrease the invasion and migration of endometrial cancer cells.