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.

5-Hydroxymethylfurfural protects against ER stress-induced apoptosis in GalN/TNF-α-injured L02 hepatocytes through regulating the PERK-eIF2α signaling pathway.

5-Hydroxymethylfurfural (5-HMF), a water-soluble compound extracted from wine-processed Fructus corni, is a novel hepatic protectant for treating acute liver injury. The present study was designed to investigate the protective effect of 5-HMF in human L02 hepatocytes injured by D-galactosamine (GalN) and tumor necrosis factor-α (TNF-α) in vitro and to explore the underlying mechanisms of action. Our results showed that 5-HMF caused significant increase in the viability of L02 cells injured by GalN/TNF-α, in accordance with a dose-dependent decrease in apoptotic cell death confirmed by morphological and flow cytometric analyses. Based on immunofluorescence and Western blot assays, we found that GalN/TNF-α induced ER stress in the cells, as indicated by the disturbance of intracellular Ca(2+) concentration, the activation of protein kinase RNA (PKR)-like ER kinase (PERK), phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α), and expression of ATF4 and CHOP proteins, which was reversed by 5-HMF pre-treatment in a dose-dependent manner. The anti-apoptotic effect of 5-HMF was further evidenced by balancing the expression of Bcl-2 family members. In addition, the knockdown of PERK suppressed the expression of phospho-PERK, phospho-eIF2α, ATF4, and CHOP, resulting in a significant decrease in cell apoptosis after the treatment with GalN/TNF-α. 5-HMF could enhance the effects of PERK knockdown, protecting the cells against the GalN/TNF-α insult. In conclusion, these findings demonstrate that 5-HMF can effectively protect GalN/TNF-α-injured L02 hepatocytes against ER stress-induced apoptosis through the regulation of the PERK-eIF2α signaling pathway, suggesting that it is a possible candidate for liver disease therapy.

[Study on fibrolytic and hypotensive activity of earthworm homogenate of ultrafiltration separation from enzymolysis].

OBJECTIVE: To study the fibrolytic and hypotensive activity of earthworm homogenate of ultrafiltration separation from simulated enzymolysis of gastrointestinal tract system. METHODS: The before and after enzymolysis homogenate of fresh earthworm was seperated with different pore size PVDF ultrafiltration membrane and its fibrolytic and hypotensive activity was assayed. RESULTS: The fibrolytic activity of the total homogenate after enzymolysis overall changed little, but decreased in the the site of higher molecular weight and increased in the lower site of molecular weight; The ACE inhibitory activity improved, especially in the filtrate of the MW 4000 membrane. CONCLUSION: The fibrolytic activity of earthworm homogenate was not reduced by the digestive simulated enzymolysis, and the retention site of MW 10 000 membrane have more fibrolytic activity; The hypotensive activity of earthworm homogenate is enhanced by the digestive simulated enzymolysis. So the stronger activity could be obtained from enzymolysis.