OBJECTIVETo investigate the effects of small interfering RNA (siRNA) targeting YAP on the proliferation and apoptosis of human periodontal ligament stem cells (hPDLSCs).

METHODSSynthesized sequences of siRNA were transfected into hPDLSCs by Lipofectamine™ 2000. The expression of YAP was identified by using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Proliferation activity was detected by using cell counting kit-8 (CCK-8). Changes in the cell cycle and apoptosis rate were detected by using flow cytometry. Results were analyzed by using SPSS 19.0, and P < 0.05 was considered statistically significant.

RESULTSExpression of YAP mRNA and protein were significantly downregulated after 48 h of transfection (P < 0.001). No obvious difference was found in the expression levels of YAP protein between 48 and 72 h, thus indicating that siRNA could inhibit the expression of YAP persistently and effectively. Proliferation activity was inhibited, and apoptosis rate was increased. Cell cycle was changed as the proportion of G₁and S phases increased (P < 0.01) and G₂ phase decreased (P < 0.05).

CONCLUSIONKnocking down YAP gene by siRNA could inhibit proliferation activity, induce apoptosis, and change the cell cycle of hPDLSCs. Thus, YAP could regulate the proliferation and apoptosis of hPDLSCs.

Adaptor Proteins, Signal Transducing ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; Humans ; Periodontal Ligament ; drug effects ; Phosphoproteins ; RNA, Messenger ; RNA, Small Interfering ; pharmacology ; Stem Cells ; drug effects ; Transfection

AIM:To investigate the effects of astragalin(AST)on activation status of astrocytes and the ex-pression level of autophagy-related proteins in the cortex of the anterior cingulate cortex of mice with a complete Freund's adjuvant(CFA)-induced inflammatory pain model.METHODS:Twenty-four 6-month-old male C57BL/6 mice were ran-domly divided into four groups:control group,saline group,CFA model group and CFA+60 mg/kg AST administration group,and six mice in each group.Mice in the AST administration group received 60 mg/kg AST by intraperitoneal injec-tion on a body weight basis for 21 d.The paw withdrawal threshold in each group of mice was evaluated by the von Frey test.The expression levels of autophagy-related factors LC3,p62,ATG12 and beclin-1,and astrocyte activation were de-tected by multiplex immunofluorescence staining in the anterior cingulate cortex of mice in each group.Western blot was used to measure the levels of autophagy-related proteins LC3,p62,ATG12 and beclin-1 in the anterior cingulate cortex of mice in each group.RESULTS:Behavioural tests showed that AST significantly increased mechanical pain thresholds in CFA mice(P＜0.05).The results from multiple immunofluorescent staining showed that AST significantly increased the fluorescence intensity of LC3(P＜0.01),ATG12(P＜0.01)and beclin-1(P＜0.05),attenuated the fluorescence intensi-ty of p62(P＜0.05),and inhibited the activation of astrocytes in the anterior cingulate cortex of CFA mice.Western blot results further confirmed that AST significantly increased the expressions of LC3(P＜0.01),ATG12(P＜0.01),beclin-1(P＜0.01),and decreased the expression of p62(P＜0.05)in the anterior cingulate cortex of CFA mice.CONCLU-SION:AST relieves CFA-induced inflammatory pain of mice,and its analgesic mechanism may be related to the inhibi-tion of activation of cortical astrocytes in the anterior cingulate cortex and the promotion of autophagy in CFA mice.