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The aim of this study was to investigate the role and mechanism of terpinen-4-ol (T4O) on high glucose (HG) -induced calcification in vascular smooth muscle cell (VSMC). To investigate the role of T4O on HG-induced calcium deposition, osteogenic phenotypic transformation and mitochondrial dynamics in VSMC, Mdivi-1, a mitochondrial dynamin-related protein 1 (Drp-1) inhibitor, was used to analyze the correlation between mitochondrial dynamics and VSMC calcification and the role of T4O. Alizarin red S staining was used to observe calcium salt deposition and flow cytometry to detect intracellular Ca2+ content; Western blot and immunofluorescence were used to detect the expression of phenotypic switching-related markers α-smooth muscle actin (α-SMA), bone morphogenetic protein 2 (BMP2) and Runt related transcription factor 2 (Runx2), and mitochondrial dynamics-related markers mitofusin 1 (MFN1), mitofusin 2 (MFN2) and Drp-1. The results showed that low and high doses of T4O could inhibit HG-induced down-regulation of α-SMA, MFN1 and MFN2 expression levels, and up-regulation of BMP2, Runx2 and Drp-1 expression levels, reduce intracellular Ca2+ content and calcium salt deposition, and effectively inhibit HG-induced VSMC calcification and mitochondrial dynamics disorders. The T4O group, Mdivi-1 group and T4O+Mdivi-1 group were able to up-regulate the expression levels of HG-induced α-SMA, MFN1 and MFN2, down-regulate the protein expression levels of BMP2, Runx2 and Drp-1, and inhibit calcium salt deposition, and there was no significant difference between the above indexes in the T4O and T4O+Mdivi-1 groups. The above findings suggest that T4O can inhibit the expression level of Drp-1, regulate the disturbance of mitochondrial dynamics, and suppress HG-induced VSMC calcification.
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Aim To investigate the effect of terpinen-4-ol (T40) on inflammatory injury of vascular smooth muscle cells (VSMCs) induced by high glucose based on the improvement of autophagic flow disorder and involved molecular signals. Methods The scratch test was used to analyze the migration ability of VSMCs, the levels of IL-1β and IL-6 in cell culture supernatant were measured by ELISA, the expression levels of inflammation-related proteins NF-κb p65, p-NF-κb p65, IL-1β, IL-18 and autophagy-related proteins p62, LC3-HYLC3-I, Beclinl, p-Beclinl were de-tected by Western blot. Results T40 inhibited migration of VSMCs induced by high glucose, reduced the secretion and release of pro-inflammatory factors IL-1β and IL-6, inhibited the expression of p-NF-κb p65/ NF-κb p65, IL-1β, IL-18, downregulated the expression of p62, LC3-TJ/LC3- I and p-Beclinl at same time. After interfering the autophagic flux of VSMCs with autophagy inhibitor chloroquine (CQ) , T40 pre-treatment significantly inhibited the protein expression levels of the above inflammatory factors and autophagy-related signals which mediated by CQ. Conclusion T40 inhibits the inflammatory injury of VSMCs induced by high glucose through improving the autophagic flow disorder.
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Aim To investigate the protective effect of cyclovirobuxine D(CVB-D) on aldosterone (ALD)-induced primary neonatal rat cardiac myocytes (PNRC-Ms) injury and the possible mechanism. Methods PNRCMs were extracted by trypsin, and the PNRCMs injury model was established by ALD (10 μmol · L
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Objective:To investigate whether ultrafine powder of Gastrodiae Rhizoma （UPG） can alleviate the learning and memory impairment of vascular dementia rats and delay the process of VD， and whether this effect is related to the release of acetylcholine （Ach） through the regulation with acetylcholinesterase （AChE） and choline acetyltransferase （ChAT） and control of cholinergic system. Method:SD rats were randomly divided into sham operation group， UPG low dose group （0.45 g·kg-1）， UPG high dose group （1.8 g·kg-1） and Huperzine A group （80 μg·kg-1）， with 12 rats in each group. The drug administration groups were given orally drugs once a day for 8 weeks， and sham group and model group were given orally the same amount of distilled water. The learning and memory ability of the rats with VD were evaluated by the Morris water maze. Htoxylin eosin（HE） staining was used for pathomorphological observation of hippocampus CA1 area of the rats. The content of Ach was determined by enzyme-linked immunosorbent assay（ELISA）， AChE and ChAT protein expressions were detected by Western blot， and expression of ChAT in hippocampus CA1 area was observed by immunohistochemistry. Result:Compared with the sham operation group， the escape latency of the model group was significantly increased （P<0.01）， and the frequency of crossings platform and the time of staying in the target quadrant were reduced significantly （P<0.01）. HE staining of hippocampal tissues from VD rat showed neuron disorders， loss and degeneration and necrosis， pyknosis of the nucleus and light coloration of the cytoplasm. The level of acetylcholine in the hippocampus was significantly decreased by ELISA （P<0.05）， the expression level of AChE protein was significantly up-regulated， and the expression level of ChAT protein was significantly down-regulated （P<0.01）. Compared with model group， each administration group could significantly reduce the escape latency of the model rats， and significantly increase the frequency of crossing platform and the time of staying in the target quadrant （P<0.01）， the content of Ach was significantly increased （P<0.05）， the expression of AChE protein was significantly down-regulated （P<0.01）， while the expression of ChAT protein was significantly up-regulated （P<0.01）. Conclusion:UPG improves the learning and memory ability of vascular dementia rats， and its mechanism may be related to the increase of Ach， ChAT level and the decrease of AChE level.
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Objective:To investigate the effect of oxymatrine (OMT) on the proliferation and migration of human colon cancer cell line HT-29 under Type Ⅱ diabetes environment by co-culturing HT-29 with insulin to simulate hyperinsulinemia. Method:The effect of OMT (2, 4, 8 mmol·L-1) on insulin-induced proliferation of HT-29 was detected by methyl thiazolyl tetrazolium (MTT) assay and cloning assay. The morphology change and cell migration were evaluated under microscope and by wound healing assay. The Annexin V/propidium iodide(PI) assay was used to detect the change of insulin-induced HT-29 cell cycle and apoptosis. Western blot was performed to validate the expression of cell cycle-related protein and cell migration protein. Result:Insulin significantly increased growth of HT-29 (P-1 showed a significant inhibitory effect in this model (P0/G1 phase (P1, CDK4 and the up-regulation of p27 by OMT might involve the growth inhibition mechanism. Furthermore, OMT reduced the migration of insulin-induced HT-29 according to wound healing assay(PPPConclusion:OMT can inhibit the proliferation and migration of insulin-induced HT-29 cells. The changes of cell cycle and migration related proteins may be correlated with the mechanism.
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<p><b>OBJECTIVE</b>To investigate the inhibitory effects of OMT on TGF-β1-induced CFBs proliferation, and then explore the mechanism.</p><p><b>METHOD</b>The experiment was randomly divided into 6 groups as following: control group (serum free DMEM), model group (20 μg x L(-1) TGF-β1), OMT low dose group (1.89 x 10(-4) mol x L(-1) + 20 μg x L(-1) TGF-β1), OMT medium dose group (3.78 x 10(-4) mol x L(-1) + 20 μg x L(-1) TGF-β1), OMT high dose group (7.56 x 10(-4) mol x L(-1) + 20 μg x L(-1) TGF-β1), SB203580 group (p38MAPK blocking agent, 1 x 10(-5) mol x L(-1) + 20 μg x L(-1) TGF-β1). Vimentin of CFBs was identified by immunocytochemical methods, α-SMA of myFBs as well. Inhibitory effects of OMT on CFBs proliferation was detected by the MTT assay. Picric acid Sirius red staining was analyzed collagen type I and collagen type III deposition. Western blot was determined the expression of p38MAPK, p-p38MAPK, collagen type I and collagen type III.</p><p><b>RESULT</b>MTT results showed that OMT significantly inhibited CFBs proliferation induced by TGF-β1 (P < 0.01) α-SMA immunocytochemical experiments suggested that OMT could protect against the CFBs proliferation. OMT could significantly decrease the deposition of collagen type I and collagen type III by Western bloting and picric acid Sirius red staining. Western blot results showed that TGF-β1 enhanced p38MAPK phosphorylation, however OMT attenuated the phosphorylation of p38MAPK induced by TGF-β1 (P < 0.01).</p><p><b>CONCLUSION</b>OMT can inhibit the CFBs proliferation induced by TGF-β1, and its mechanism may be involved in inhibiting p38MAPK phosphorylation.</p>