D-阿洛糖对OGD/R诱导的HT22细胞损伤及Gal-3表达的影响

doi:10.3877/cma.j.issn.2095-9141.2023.03.002

目的

探讨D-阿洛糖处理对氧糖剥夺再灌注（OGD/R）诱导的HT22细胞损伤及半乳糖凝集素-3（Gal-3）表达的影响。

方法

正常培养的HT22细胞，传代贴壁后，随机分为对照组、OGD/R组、D-阿洛糖组与抑制剂组。构建OGD/R模型，设立OGD/R组。D-阿洛糖组：构建OGD/R模型，复氧复糖时于培养基中加入10 mmol/L D-阿洛糖，持续到复氧复糖结束。抑制剂组：构建OGD/R模型，复氧复糖时于培养基中同时加入D-阿洛糖和TD139，持续到复氧复糖结束。采用CCK8法检测细胞存活率；Western blot检测Gal-3以及凋亡相关蛋白Bax、Bcl-2、Caspase-3、Cleaved caspase-3的表达；采用流式细胞术检测细胞凋亡。

结果

与OGD/R组相比，D-阿洛糖组细胞存活率显著升高，细胞凋亡率显著降低，Bax、Cleaved caspase-3、Gal-3、LGALS3BP蛋白表达量显著降低，Bcl-2蛋白表达量显著升高，差异均有统计学意义（P<0.05）；而Caspase-3蛋白表达量比较差异无统计学意义（P>0.05）。与D-阿洛糖组相比，抑制剂组的Bcl-2/Bax表达显著增加，Cleaved caspase-3蛋白表达量显著减少，差异均有统计学意义（P<0.05）；Caspase-3蛋白表达量比较差异无统计学意义（P>0.05）。

结论

D-阿洛糖处理对OGD/R诱导的HT22细胞损伤有保护作用以及D-阿洛糖可能通过抑制Gal-3发挥保护作用。

关键词: HT22细胞, 氧糖剥夺再灌注, D-阿洛糖, 半乳糖凝集素-3

Objective

To investigate the effects of D-allose treatment on oxygen glucose deprivation/reperfusion (OGD/R) induced HT22 cell damage and galactin-3 (Gal-3) expression.

Methods

HT22 cells in normal culture were randomly divided into control group, OGD/R group, D-allose group and inhibitor group after passage and adhesion. OGD/R model was constructed and set it as the OGD/R group. D-allose group: OGD/R model was constructed, 10 mmol/L D-allose was added into the medium during the reoxygenation and reglucose and continued until the end of the reoxygenation and reglucose. Inhibitor group: OGD/R model was constructed, and D-allose and TD139 were added to the culture medium at the same time during reoxygenation and disaccharide, which lasted until the end of reoxygenation and disaccharide. CCK8 assay was used to detect cell survival rate. The expression of Gal-3 and apoptosis-related proteins Bax, Bcl-2, Caspase-3, Cleaved caspase-3 were detected by Western blot. Apoptosis was detected by flow cytometry.

Results

Compared with OGD/R group, the survival rate of cells in D-allose group was significantly increased, the apoptosis rate was significantly decreased, the protein expression levels of Bax, Cleaved caspase-3, Gal-3 and LGALS3BP were significantly decreased, and the expression level of Bcl-2 protein was significantly increased, with statistical significance (P<0.05); but there was no significant difference in Caspase-3 protein expression (P>0.05). Compared with the D-allose group, the expression of Bcl-2/Bax in inhibitor group was significantly increased, Cleaved caspase-3 protein expression was significantly decreased, with statistical significance (P<0.05); there was no significant difference in Caspase-3 protein expression (P>0.05).

Conclusion

D-allose treatment has a protective effect on OGD/R-induced HT22 cell damage, and D-allose may play a protective role by inhibiting Gal-3.

Key words: HT22 cell, Oxygen glucose deprivation/reperfusion, D-allose, Galactin-3

图1 OGD/R的处理时间对HT22细胞存活率的影响（n=6）与对照组比较，^aP<0.05；OGD/R：氧糖剥夺再灌注

Fig.1 Effects of OGD/R processing time on the survival rate of HT22 cells (n=6)

图2 OGD/R的处理时间对HT22细胞中Gal-3蛋白表达的影响（n=3）A：Gal-3和β-actin蛋白表达量；B：Gal-3和β-actin相对表达水平；与对照组比较，^aP<0.05；OGD/R：氧糖剥夺再灌注；Gal-3：半乳糖凝集素-3

Fig.2 Effects of OGD/R processing time on Gal-3 protein expression in HT22 cells (n=3)

图3 不同浓度的D-阿洛糖对OGD/R后细胞存活率的影响（n=6）与对照组相比，^aP<0.05；与OGD/R组相比，^bP<0.05；OGD/R：氧糖剥夺再灌注

Fig.3 Effect of different concentrations of D-allose on cell survival after OGD/R (n=6)

图4 D-阿洛糖对OGD/R后细胞凋亡的影响（n=3）A：各组流式检测细胞凋亡象限图（四个象限的意义：B1：坏死细胞；B2：晚期凋亡细胞；B3：未发生凋亡的细胞；B4：早期凋亡细胞）；B：流式检测细胞凋亡统计图；与对照组比较，^aP<0.05；与OGD/R组比较，^bP<0.05；OGD/R：氧糖剥夺再灌注

Fig.4 Effect of D-allose on apoptosis after OGD/R (n=3)

图5 D-阿洛糖对OGD/R后细胞凋亡相关蛋白表达的影响（n=3）A：Bax、Caspase-3、Cleaved caspase-3和β-actin蛋白表达量；B：Bax和β-actin相对表达水平；C：Caspase-3和β-actin相对表达水平；D：Cleaved caspase-3和β-actin相对表达水平；与对照组比较，^aP<0.05；与OGD/R组比较，^bP<0.05；OGD/R：氧糖剥夺再灌注

Fig.5 Effect of D-allose on apoptosis-related protein expression after OGD/R (n=3)

图6 D-阿洛糖对OGD/R后Gal-3及下游蛋白表达的影响（n=3）A：Gal-3、LGALS3BP、Bcl-2和β-actin蛋白表达量；B：Gal-3和β-actin相对表达水平；C：LGALS3BP和β-actin相对表达水平；D：Bcl-2和β-actin相对表达水平；与对照组比较，^aP<0.05；与OGD/R组比较，^bP<0.05；OGD/R：氧糖剥夺再灌注；Gal-3：半乳糖凝集素-3

Fig.6 Effect of D-allose on expression of Gal-3 and downstream protein after OGD/R (n=3)

图7 TD139对OGD/R后细胞凋亡相关蛋白表达的影响（n=3）A：Bax、Bcl-2、Caspase-3、Cleaved caspase-3和β-actin蛋白表达量；B：Bcl-2和β-actin相对表达水平/Bax和β-actin相对表达水平；C：Caspase-3和β-actin相对表达水平；D：Cleaved caspase-3和β-actin相对表达水平；与对照组比较，^aP<0.05；与OGD/R组比较，^bP<0.05；与D-阿洛糖组比较，^cP<0.05；OGD/R：氧糖剥夺再灌注

Fig.7 Effect of TD139 on apoptosis-related protein expression after OGD/R (n=3)

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Effects of D-allose on OGD/R-induced HT22 cell damage and Gal-3 expression

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Effects of D-allose on OGD/R-induced HT22 cell damage and Gal-3 expression