赣南医科大学学报2025，Vol.45Issue(11)：1027-1032,1068,7.DOI:10.3969/j.issn.2097-7174.2025.11.001
小檗碱杀伤ST3芽囊原虫的作用研究
Investigation of the insecticidal activity of berberine against ST3 subtype Blastocystis
摘要
Abstract
Objective:To explore the killing activity of berberine against ST3 subtype Blastocystis and its cytotoxicity on T84 cells,and to analyze its molecular regulatory mechanism on the expression of inflammatory factors.Methods:⑴The concentration of ST3 subtype Blastocystis was adjusted to 1×106·mL-1 and the counts were taken at 24,48,72,96 and 120 h of culture,respectively;⑵ The negative control group(PBS),positive control group(MNZ100),200 μmol·L-1 berberine group(BBR200),400 μmol·L-1 berberine group(BBR400),600 μmol·L-1 berberine group(BBR600),800 μmol·L-1 berberine group(BBR800)and 1 000 μmol·L-1 berberine group(BBR1 000)were set up.The parasites in each group were cultured in an anaerobic incubator at 37℃,and the parasite counts were determined at 24,72 and 144 h using a hemocytometer;⑶ The cells were divided into blank control group(PBS),parasite control group(BH),BBR200 group(BBR200),and BBR400 group(BBR400).After 24 h of culture,the morphological changes of cells in each group were observed under an inverted microscope;⑷ The activity of T84 cells was detected by CCK-8 assay;⑸The mRNA transcription levels of pro-inflammatory factors MCP-1,TNF-α,GM-CSF and anti-inflammatory factor TGF-β in T84 cells were determined by RT-qPCR;⑹ The protein expression levels of pro-inflammatory factor MCP-1 and anti-inflammatory factor TGF-β were measured by ELISA.Results:⑴ The ST3 strain was in the logarithmic growth phase from 24 to 72 h,reached the stationary phase at 72 to 96 h,and then gradually entered the decline phase,with the number of parasites decreasing progressively;⑵ The number of parasites in the PBS group increased by approximately 2 times within 72 h;the inhibition rate of the MNZ100 group was 80%at 72 h and 95%at 144 h;the inhibition rates of the BBR200,BBR400,BBR600,BBR800,and BBR1 000 groups were 49.2%,75%,84.2%,94%,and 98.4%at 72 h,respectively,and increased to 74%,91.2%,96.6%,98.2%,and 99.2%at 144 h.The results indicated that berberine had a concentration-dependent inhibitory effect on ST3 subtype Blastocystis,with a significant inhibitory effect on the proliferation of ST3 subtype Blastocystis at 200 μmol·L-1,and the inhibitory effect was close to that of metronidazole at 400 μmol·L-1,and better than that of metronidazole at 600 μmol·L-1;⑶ After 144 h of culture,the cells in the PBS group were morphologically intact,with clear cell membranes and full contours.The cells in the BBR400 group showed typical apoptotic features such as cell volume expansion,cell membrane rupture,cytoplasmic leakage,and nuclear condensation with deep staining.The morphological changes in the MNZ100 group were similar to those in the BBR400 group,but the degree of cell swelling was more significant;⑷ The cells in the PBS group and the BBR400 group showed typical cobblestone-like adherent growth characteristics.Some cells in the BBR200 group showed rounding and detachment.In the BH group,extensive cell rounding and detachment occurred;⑸ The cell viability in the BH group was significantly lower than that in the PBS group(P＜0.01).The cell viability in the BBR200 and BBR400 groups was significantly higher than that in the BH group(P＜0.01),and there was a positive correlation between berberine concentration and cell viability(r=0.92,P＜0.05);⑹ The RT-qPCR results showed that compared with the PBS group,the mRNA expression of MCP-1,TNF-α,and GM-CSF increased,and the mRNA expression of TGF-β decreased in the BH group,with statistically significant differences(P＜0.05).Compared with the BH group,the mRNA expression of MCP-1,TNF-α,and GM-CSF decreased in the BBR200 group,with statistically significant differences(P＜0.05),and the mRNA expression of TGF-β increased,but the difference was not statistically significant(P＞0.05);⑺ The ELISA results showed that compared with the PBS group,the protein expression of MCP-1 increased and the protein expression of TGF-β decreased in the BH group,with statistically significant differences(P＜0.05).Compared with the BH group,the protein expression of MCP-1 decreased in the BBR200 group,with statistically significant differences(P＜0.05),and the protein expression of TGF-β increased,but the difference was not statistically significant(P＞0.05).Conclusion:Berberine has a significant concentration-dependent killing activity against ST3 subtype Blastocystis,effectively inhibiting the proliferation of the parasite and inducing morphological changes.ST3 subtype Blastocystis exerts a definite toxic effect on T84 cells by up-regulating pro-inflammatory factors and down-regulating anti-inflammatory factors,while berberine exerts an insecticidal effect by reversing the imbalance of these inflammatory factors and reducing the toxic effects.关键词
芽囊原虫/小檗碱/炎症因子/细胞毒性/浓度依赖性效应Key words
Blastocystis/Berberine/Inflammatory factors/Cytotoxicity/Concentration-dependent effects分类
医药卫生引用本文复制引用
ZHONG Yue,YAN Yi-ming,XIE Fu-hua..小檗碱杀伤ST3芽囊原虫的作用研究[J].赣南医科大学学报,2025,45(11):1027-1032,1068,7.基金项目
国家自然科学基金项目(81260255) （81260255）
江西省教育厅科学技术研究项目(GJJ211546) （GJJ211546）
