AIM:To investigate the effect of 18 alpha-glycyrrhetinic acid (18α-GA) on delaying the senescent progress and promoting the proliferation in late-passage bone marrow mesenchymal stem cells ( BMSCs ) .METHODS:Late-passage BMSCs were incubated with 2.0 mg /L 18α-GA or the same volume of DMSO for 30 d, and the cells were harvested to determine the proteasome activity .The expression of senescence-related proteins p53, p21 and p16 was detec-ted by senescence-associated β-galactosidase ( SA-β-Gal) staining and Western blot .The cell proliferation , the expression level of cell cycle-related proteins and cell cycle distribution of the cells were measured by CCK -8 assay, BrdU incorpora-tion, Western blot and flow cytometry.RESULTS:Compared with DMSO group, the proteasome activity in 18α-GA group increased significantly by about 0.2 times (P<0.01).SA-β-Gal-positive cells in 18α-GA group decreased, and cell stai-ning was lighter.The contents of p53 and p21 in 18α-GA group were decreased (P<0.05).The results of CCK-8 assay showed that the A value in 18α-GA group was 0.3 times higher than that in DMSO group (P<0.01).BrdU incorporation showed the increased proliferation in 18α-GA group compared with DMSO group ( P<0.05).The cells in G1 phase in 18α-GA group decreased significantly compared with DMSO group , while the cells in S phase increased significantly ( P<0.05).The expression level of cyclin D1 in 18α-GA group was 2.8 times higher than that in DMSO group (P<0.01), and the CDK4 level was 1.4 times higher than that in DMSO group (P<0.05).CONCLUSION:Activation of the pro-teasome activity by 18α-GA delays the aging process in the BMSCs and promotes the cell proliferation via up -regulation of the cell cycle-related proteins .

AIM:To investigate the effect of tert-butylhydroquinone ( tBHQ) on the replicative senescence of bone marrow mesenchymal stem cells (BMSCs).METHODS: Late stage BMSCs were continuously treated with tBHQ at concentration of 30 μmol/L for 4 weeks and the cells were used for the following assays immediately .The proteasomal ac-tivity was determined by chemiluminescence method .The samples were subjected to CCK-8 assay and BrdU incorporation as well as flow cytometry analysis for analyzing the cell vitality and proliferation .Percentage of senescent cells was detected by senescence-associatedβ-galactosidase ( SA-β-Gal) staining.The expression of P53 was measured by Western blot .RE-SULTS:After the continuous treatment of tBHQ (30 μmol/L) for 4 weeks, the proteasomal activity of late stage BMSCs increased by 21.96%±1.98%(P<0.05).The cell vitality and survival were significantly increased with the increases in tBHQ doses till 40 μmol/L, and no cytotoxicity reaction with the increased dose of tBHQ till 120 μmol/L was observed . BrdU-positive cells, which represented the cell proliferation , were significantly increased (P<0.05).The proliferation in-dex was also significantly increased by flow cytometry analysis (P<0.05).The SA-β-Gal positive cells and the expression of P53 were decreased (P<0.05).CONCLUSION:tBHQ delays the proteasome dysfunction associated senescence pro-gress of BMSCs by increasing the proteasomal activity .

Objective:To investigate the expressions and clinical significance of tetraspanin CO-029 and integrin αv in intrahepatic cholangiocarcinoma (ICC ).Methods:Tissue microarray (TMA) was used to detect the expression of CO-029 and αv in 254 cases of intrahepatic cholangiocarcinoma. The relationship between the two factors and clinicopathological features, recurrence, metastasis and prognosis was analyzed.Spearman method was used to analyze their correlation.Relationship between αv and CO-029 was studied by mass spectrometry and database search,immunoprecipitation and Western blot were used to detect the coexistence.Results:Tissue microarray analysis showed that the positive expression rate of CO-029 was 51.6% (131/254), and the positive expression rate of αv was 61.4% (156/254). The expression of CO-029 and αv were closely correlated with tumor envelope, size, number and TNM stage ( P<0.05). According to the time of recurrence (TTR), the expressions of CO-029 and αv in early postoperative recurrence group (TTR <1 year) were significantly higher than those in non recurrence group (TTR ≥ 1 year). The patients with high CO-029 expression were more likely to relapse ( HR=2.01, 95% CI=1.45-2.79; P<0.001) and had shorter survival time ( HR=2.03, 95% CI=1.46-2.81; P<0.001). The patients with high expression of αv had shorter recurrence time ( HR=1.85, 95% CI=1.38-2.47; P<0.001) and shorter survival time ( HR=1.95, 95% CI=1.40-2.71; P<0.001). Co immunoprecipitation and Western blot confirmed that αv and CO-029 formed a complex. There was a positive correlation between CO-029 and αv in intrahepatic cholangiocarcinoma ( r=0.401, P<0.01). Conclusions:The differential expression of CO-029 and αv were closely related to the recurrence, metastasis and prognosis of intrahepatic cholangiocarcinoma, and CO-029 may couple with αv to form a complex to promote the invasion and metastasis of intrahepatic cholangiocarcinoma.

Focal cortical dysplasia (FCD) is one of the most common causes of drug-resistant epilepsy. Dysmorphic neurons are the major histopathological feature of type II FCD, but their role in seizure genesis in FCD is unclear. Here we performed whole-cell patch-clamp recording and morphological reconstruction of cortical principal neurons in postsurgical brain tissue from drug-resistant epilepsy patients. Quantitative analyses revealed distinct morphological and electrophysiological characteristics of the upper layer dysmorphic neurons in type II FCD, including an enlarged soma, aberrant dendritic arbors, increased current injection for rheobase action potential firing, and reduced action potential firing frequency. Intriguingly, the upper layer dysmorphic neurons received decreased glutamatergic and increased GABAergic synaptic inputs that were coupled with upregulation of the Na⁺-K⁺-Cl^- cotransporter. In addition, we found a depolarizing shift of the GABA reversal potential in the CamKII-cre::PTEN^flox/flox mouse model of drug-resistant epilepsy, suggesting that enhanced GABAergic inputs might depolarize dysmorphic neurons. Thus, imbalance of synaptic excitation and inhibition of dysmorphic neurons may contribute to seizure genesis in type II FCD.
Animals ; Drug Resistant Epilepsy/surgery* ; Epilepsy/pathology* ; Malformations of Cortical Development/pathology* ; Malformations of Cortical Development, Group I ; Mice ; Neurons/pathology* ; Seizures/pathology*