Puerarin promotes the viability and differentiation of MC3T3-E1 cells by enhancing LC3B-mediated autophagy through downregulation of miR-204.

Puerarin is a bioactive substance extracted from Pueraria lobata. It is known to promote the viability, differentiation and mineralization of osteoblasts. However, the molecular mechanisms involved in these activities are not well understood. The present study was conducted with the aim of elucidating the effect of puerarin on osteoblasts and to explore the underlying mechanism. CCK-8 analysis showed that puerarin (0.1, 1 and 10 µM) promoted the viability of osteoblastic MC3T3-E1 cells, with 1 µM of puerarin exhibiting the strongest effect. Moreover, 1 µM puerarin significantly increased the activity of alkaline phosphatase (ALP) and the formation of mineralized nodules in the MC3T3-E1 cells. Treatment with 1 µM puerarin for 72 h led to a significant upregulation in the expression level of microtubule-associated light chain 3 (LC3)B and Beclin1 proteins. This treatment was more effective in promoting LC3B expression than what was observed following treatment with rapamycin (overexpression for autophagy). The bilayer membrane structure of autophagosomes was observed by electron microscopy. Conversely, 3-methyladenine (3-MA, inhibitor of autophagy) reduced the cell viability as well as the activity of alkaline phosphatase (ALP) in MC3T3-E1 cells, although, there was no significant influence on mineralization. Prediction results of the biological information showed that LC3B could be a direct target of microRNA-204 (miR-204). In the present study, the expression level of miR-204 was decreased by puerarin. miR-204 mimics significantly decreased LC3B expression and inhibited auotophagosome formation, while the miR-204 inhibitor had the opposite effects. To conclude, the results of the present study suggest that puerarin promotes the viability and differentiation of MC3T3-E1 cells through autophagy, which is possibly associated with miR-204-regulated LC3B upregulation.

Puerarin promotes the viability and differentiation of MC3T3­E1 cells by miR­204­regulated Runx2 upregulation.

Puerarin has attracted increasing attention because of its beneficial effects on anti­osteoporosis, but the molecular mechanisms underlying its actions on osteoblasts are not fully understood. The current study aimed to investigate the effect of puerarin on the cell viability and differentiation of mouse MC3T3­E1 osteoblast­like cells in vitro and its underlying mechanisms. The results indicated that 0.01, 0.1 and 1 mg/ml puerarin significantly promoted the viability of osteoblasts, enhanced alkaline phosphatase (ALP) activity and increased the expression of transforming growth factor­ß1, Smad2, Smad3 and Runt­related transcription factor (Runx)2. Micro (mi)RNA target prediction programs predicted that miR­204 may directly target Runx2. Following treatment with 0.1 mg/ml puerarin for 48 h, the expression level of miR­204 was downregulated. Besides, miR­204 dramatically repressed the luciferase activity of wildtype Runx2 3'­UTR transfected cells, but not that of the mutant ones. Overexpression of miR­204 in osteoblasts significantly decreased the protein expression of Runx2, while inhibition of miR­204 enhanced Runx2's expression. In addition, overexpression of miR­204 inhibited the cell viability and ALP activity of osteoblasts, while inhibition of miR­204 had the opposite effect. The results suggested that puerarin may promote MC3T3­E1 osteoblast­like cell viability and differentiation, which may be related to the downregulation of miR­204 and the following activation of Runx2.

Daucosterol protects neurons against oxygen-glucose deprivation/reperfusion-mediated injury by activating IGF1 signaling pathway.

We previously reported that daucosterol (a sterolin) up-regulates the expression of insulin-like growth factor I (IGF1)(1) protein in neural stem cells. In this study, we investigated the effects of daucosterol on the survival of cultured cortical neurons after neurons were subjected to oxygen and glucose deprivation and simulated reperfusion (OGD/R)(2), and determined the corresponding molecular mechanism. The results showed that post-treatment of daucosterol significantly reduced neuronal loss, as well as apoptotic rate and caspase-3 activity, displaying the neuroprotective activity. We also found that daucosterol increased the expression level of IGF1 protein, diminished the down-regulation of p-AKT(3) and p-GSK-3ß(4), thus activating the AKT(5) signal pathway. Additionally, it diminished the down-regulation of the anti-apoptotic proteins Mcl-1(6) and Bcl-2(7), and decreased the expression level of the pro-apoptotic protein Bax(8), thus raising the ratio of Bcl-2/Bax. The neuroprotective effect of daucosterol was inhibited in the presence of picropodophyllin (PPP)(9), the inhibitor of insulin-like growth factor I receptors (IGF1R)(10). Our study provided information about daucosterol as an efficient and inexpensive neuroprotectants, to which the IGF1-like activity of daucosterol contributes. Daucosterol could be potentially developed as a medicine for ischemic stroke treatment.

Discrepancy between the effects of morronside on apoptosis in human embryonic lung fibroblast cells and lung cancer A549 cells.

Morroniside is a water-soluble compound extracted from the fruit of Cornus officinalis and is used to protect lung activity against aging. In the present study, the manner in which morroniside regulates normal lung and cancer cells was examined. The human embryonic lung fibroblast (HELF) cell line and lung cancer A549 cell line, and their responses to morroniside treatment, were examined. Results showed that morroniside reverses the apoptotic effect of H2O2 on HELF cell growth, protecting cell proliferation and normal cell morphology and inhibiting apoptosis. However, these effects were not present in A549 cells. Western blotting showed that morroniside also markedly downregulated retinoblastoma protein in HELF cells. These results suggest that morroniside treatment exhibits different effects on apoptosis in HELF and A549 cells, making it a viable compound for decreasing the side effects of anticancer medicines in normal cells.

[Study on synergistic effect of sodium cantharidinate combined with chemotherapeutic drugs on hepatic carcinoma and its effective mechanism].

OBJECTIVE: To study the synergistic effect on hepatoma cell(SMMC-7721) and the reduction killing effect on normal liver cells(LO-2) treated with sodium cantharidinate (SCA) in combination with fluorouracil(5-FU) or cisplatin(DDP) as well as the related mechanism. METHODS: MTT assay was used to select the best ratio of SCA with 5-FU or SCA with DDP which had less toxicity on LO-2 cell line and had synergistic effect on SMMC-7721 cell line; Flow cytometry assay was used to analyze the apoptosis-induction of the different ratio of drugs on both cell lines; Hoechst-33258 fluorescent staining assay was used to observe the nuclear morphological changes of cells; Immunoblotting assay was used to analyze the Ras/Raf/ERK1/2 signaling pathway and the apoptosis related signaling pathway in both cell lines. RESULTS: MTI assay indicated that the proliferation inhibition of SCA,5-FU and DDP on SMMC-7721 cell line was in a time-and dose-dependent manner respectively. Among them, SCA had a more significant inhibition on SMMC-7721 cell line than on LO-2 after 12 h or 24 h treatment (P <0. 01). Moreover, after a treatment of 48 h,the ratio of 2. 5 µg/mL SCA and 2 µg/mL DDP showed a more significant inhibition on SMMC-7721 cell line than on LO-2 cell line,which was then be considered as the optimal concentration ratio for the following experiment. Co-treatment of SCA (2. 5 µg/mL) with DDP (2 µg/mL) induced a more significant apoptosis on SMMC-7721 cell line compared with single treatment with SCA (2. 5 µg/mL) or DDP (2 µg/mL) respectively (P < 0. 01). After a 48 h treatment of the optimal ratio of drugs, the significant morphological apoptotic characteristics were observed both under inverted microscope and by Hoechst-33258 fluorescent staining assay in both cell lines. The results of Western blot assay showed that this ratio of drugs could significantly increase the protein expression of Bax,P53 and P21 and decreased the expression of BCL-2, Casepase-3, p-Erk, p-Ras and p-c-Raf in SMMC-7721 cells. Meanwhile,the effect on the proteins mentioned above was lesser in LO-2 cells. CONCLUSION: These results indicates that 2. 5 µg/mL SCA + 2 µg/mL DDP showed a higher inhibition on the hepatic carcinoma cells and a relatively lower cytotoxicity on normal liver cells. The major anti-cancer mechanism is related with the inhibition on Erk signaling pathway and the induction of apoptosis through the mitochondrial pathway.

Daucosterol promotes the proliferation of neural stem cells.

Neural stem cells (NSCs) are self-regenerating cells, but their regenerative capacity is limited. The present study was conducted to investigate the effect of daucosterol (a sterolin) on the promotion of NSC proliferation and determine the corresponding molecular mechanism. Results of cell counting kit-8 (CCK-8) assay showed that daucosterol significantly increased the quantity of viable cells and the effectiveness of daucosterol was similar to that of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Flow cytometry detection of CFSE-labeled (CFSE, carboxyfluorescein diacetate succinimidyl ester) NSCs showed that Div Index (or the average number of cell divisions) and % Divided (or the percentage of cells that divided at least once) of the cells were increased, indicating that daucosterol increased the percentage of NSCs re-entering the cell cycle. mRNA microarray analysis showed that 333 genes that are mostly involved in the mitotic cell cycle were up-regulated. By contrast, 627 genes that are mostly involved in differentiation were down-regulated. In particular, insulin-like growth factor I (IGF1) was considered as an important regulatory gene that functionally promoted NSC proliferation, and the increased expression of IGF1 protein was validated by ELISA. In addition, the phosphorylation of AKT was increased, indicating that the proliferation-enhancing activity of daucosterol may be involved in IGF1-AKT pathway. Our study provided information about daucosterol as an efficient and inexpensive growth factor alternative that could be used in clinical medicine and research applications.

Microarray Analysis of mRNA and MicroRNA Expression Profile Reveals the Role of ß -Sitosterol-D-glucoside in the Proliferation of Neural Stem Cell.

Neural stem cells (NSCs) are self-regenerating cells, but their regenerative capacity is limited. The present study was conducted to investigate the effect of ß -sitosterol-D-glucoside (BSSG) on the proliferation of hippocampal NSCs and to determine the corresponding molecular mechanism. Results of CCK-8 assay showed that BSSG significantly increased NSC proliferation and the effectiveness of BSSG was similar to that of basic fibroblast growth factor and epidermal growth factor. mRNA expression profiling showed that 960 genes were differentially expressed after NSCs were treated with BSSG. Among the 960 genes, IGF1 is considered as a key regulatory gene that functionally promotes NSC proliferation. MicroRNA (miRNA) expression profiling indicated that 30 and 84 miRNAs were upregulated and downregulated, respectively. miRNA-mRNA relevance analysis revealed that numerous mRNAs including IGF1 mRNA were negatively regulated by miRNAs with decreased expression, thereby increasing the corresponding mRNA expression. The increased expression of IGF1 protein was validated by ELISA. Picropodophyllin (PPP, an inhibitor of IGF-1R) inhibition test confirmed that the proliferation-enhancing effect depended on IGF1. This study provided information about BSSG as an efficient and inexpensive growth factor alternative, of which the effect is closely involved in IGF1.

[Effect of 5-hydroxymethyl furfural on BCL-2 and NF-kappaB gene expression of apoptotic rat hippocampal neurons injured by H2O2].

OBJECTIVE: To investigate the effect of 5-hydroxymethyl furfural (5-HMF) on apoptosis and BCL-2, NF-kappaB gene expression of rat hippocampal neurons injured by hydroperoxide (H2O2). METHODS: Hippocampal neurons of newly born rat were cultured in vivo and injured by H2O2. Effect of different concentration of 5-HMF on cell viability was measured by MTT. Flow cytometer (FCM) was used to measure the apoptosis of rat hippocampal neurons pre-cultured with different concentration of 5-HMF,Western blotting was used to measure the expression of BCL-2 and NF-kappaB gene. RESULTS: It revealed that the high and medium dosage of 5-HMF could increase the activity of rat hippocampal. The high, medium and low dosage of 5-HMF also increased the expression of BCL-2 gene and decreased the expression of NF-kappaB gene. CONCLUSION: 5-HMF could restrain the apoptosis of cultured hippocampal neurons injured by H2O2. The mechanism may be related to increasing in BCL-2 level and decreasing in NF-kappaB level.

Investigation on the morphological protective effect of 5-hydroxymethylfurfural extracted from wine-processed Fructus corni on human L02 hepatocytes.

AIM: To determine the mode of action of 5-hydroxymethylfurfural (5-HMF) extracted from wine-processed Fructus corni on hepatoprotective activities, the effects of 5-HMF on H(2)O(2)-induced human L02 hepatocytes injury was examined. MTHODS: Hepatocytes L02 injured by H(2)O(2) was treated by 5-HMF. The morphological changes of the cells were observed under inverted phase-contrast, fluorescence, and transmission electron microscopy and the activities of caspase-9 and caspase-3 were tested by enzyme-linked immunosorbent detector. RESULTS: It revealed that 5-HMF improved the morphology of H(2)O(2)-treated human L02 hepatocytes, and also inhibited the level of caspase-9 and caspase-3 of them. CONCLUSIONS: These results suggested a morphological hepatocyte protective effect and the anti-apoptosis mechanism by 5-HMF.