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Objective To investigate the influence mechanism of Cordyceps sinensis (CS) on β-glycerophosphate-induced vascular smooth muscle cell (VSMC) calcification.Methods The effect of CS on VSMC cell viability was detected by CCK-8.The cellular models of rat VSMC calcification were established by treating with β-glycerophosphate (β-GP,10 mmol/L);then CS (10 mg/L),autophagy inhibitor 3-methyladenine (3-MA,5 mmol/L),and AMPK inhibitor compound C (CC,10 μmol/L) were added to the cell cultures.There were a total of 5 experiment groups:VSMC cultured in normal medium (Control),VSMC treated with β-GP,VSMC treated with β-GP and CS,VSMC treated with 3-MA,β-GP and CS,and VSMC treated with CC,β-GP and CS.The calcium nodules and calcium content were examined with alizarin red S staining and the O-cresolphthaleincomplexone method,respectively.The autophagosomes within the VSMC were observed using transmission electron microscope (TEM).Immunofluorescence showed the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta.In addition,levels of osteogenic related proteins,autophagy related proteins,and AMPK/mTOR pathway related proteins were evaluated by Western blotting.Results CS increased the number of autophagosomes and the accumulation of LC3 puncta within VSMC.It also upregulated the protein levels of LC3 Ⅱ/LC3 Ⅰ,beclin1,α-SMA,and p-AMPK;whereas,the protein levels of Runx2 and p-mTOR,as well as calcium nodules and calcium content were reduced (all P ＜ 0.01).When the cells were pretreated with 3-MA before treating with β-GP and CS,the autophagosomes,accumulation of LC3 puncta,and protein levels of LC3 Ⅱ/LC3 Ⅰ,beclinl,and α-SMA were decreased (all P ＜ 0.01);however,the protein level of Runx2,and the calcium nodules and calcium content were increased (all P ＜ 0.01).Nevertheless,when the cells were pretreated with CC before giving β-GP and CS,the autophagosomes,the accumulation of LC3 puncta,and the expression levels of p-AMPK,LC3 Ⅱ/LC3 Ⅰ,beclin1,and α-SMA were significantly down-regulated (all P ＜ 0.01);whereas,the expression levels of Runx2 and p-mTOR,as well as calcium nodules and calcium content were increased (all P ＜ 0.01).Conclusions CS can effectively alleviate β-GP-induced VSMC calcification,which may be due to the activation of autophagy by AMPK/mTOR signaling pathway.
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Objective@#To investigate the role and mechanism of Hydroxysafflor yellow A (HSYA) in the calcification of vascular smooth muscle cells (VSMC) induced by β-glycerol phosphate (β-GP).@*Methods@#VSMC were cultured with 10% fetal bovine serum+1% double anti-high glucose DMEM medium at 37℃ and 5%CO2 incubator, and were subcultured according to cell growth density at 1∶4 ratio. The cells were divided into three groups: control group (NC), high-phosphate-induced calcification (HP) group, and HSYA intervention (HSYA) group. The Calcium deposition amount was measured by alizarin red staining and calcium determination kit. The expressions of ALP, RUNX2, RANKL, α-SMA and inflammation indicators TLR4, TNF-α, IL-8 were detected by Western blotting method; Western blotting was also used to detect calcification index alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2). Nuclear factor kappa B receptor activating factor ligand(RANKL), α-smooth muscle actin (α-SMA), and the expressions of TLR4/NF-κB pathway and inflammatory response-related indicators Toll-like receptor 4 (TLR4), interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α). The nuclear protein and cytoplasmic proteins were respectively extracted. The expressions of p65 in nucleus and cytoplasm, as well as the expressions of p65 and phosphorylated p65 in total proteins were detected by Western blotting method. Superoxide dismutase (SOD) and malondialdehyde (MDA) kit were used to detect the content of antioxidant enzymes and oxidation end products in cells.@*Results@#Western blotting showed that the expressions of ALP, RUNX2 and RANKL in HSYA group were significantly lower than that in HP group. The expression of α-SMA was increased than that of HP group (all P<0.01). The expression levels of TLR4, TNF-α, IL-8 and p-NF-κB/p65 in HSYA group were decreased compared with that in the HP group, and p65 was decreased in nucleus and increased in cytoplasm (all P<0.05). SOD content in HSYA group was significantly higher than that in HP group, and MDA content was significantly lower than that in HP group (all P<0.01).@*Conclusions@#HSYA can reduce calcification and calcium deposition of vascular smooth muscle cells induced by high phosphorus. The mechanism is related to the inhibition of the activation of TLR4/NF-κB pathway and the inhibition of oxidative stress.
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Objective To investigate the possible mechanism of sclerostin/Lrp4 in calcification of VSMC induced by high phosphorus and the protective effect of Ginkgo biloba extract.Methods Aortic vascular smooth muscle cells (VSMCs) of SD rats were extracted and identified.VSMCs were divided into normal control group,high phosphorus induced calcification group (10 mmol/L β-glycerophosphate+50 μg/ml ascorbic acid),and high phosphorus induced calcification+Ginkgo biloba extract intervention group (10 mmol/L β-glycerophosphate+50 μg/ml ascorbic acid+0.5 mg/ml GBE),cultured in different mediums for 14 days.Vonkossa staining and alizarin red staining were used to detect the calcification of VSMCs.The mRNA level of BGP was detected by real time PCR,and the protein expressions of sclerostin and Lrp4 were detected by Western blot.Results Compared with normal control group,vonkossa staining and alizarin red staining showed significant calcium deposition in calcification group.Compared with calcification group,calcium salt deposition was significantly reduced in GBE treatment group.Real time PCR results showed β-catenin and BGP mRNA expressions in VSMC calcification group were higher than those in normal control group (P＜ 0.05).mRNA expressions of β-catenin and BGP in GBE treatment group were lower than those in calcification group (all P ＜ 0.05).Compared with normal control group,the protein expression of sclerostin was increased,but the protein expression of Lrp4 was decreased in calcified group (all P ＜ 0.05).Compared with calcification group,the protein expression of sclerostin decreased and the protein expression of Lrp4 increased in GBE treatment group (all P ＜ 0.05).Conclusions High phosphorus can induce VSMC calcification by activating Wn/β-catenin signaling pathway.Sclerostin/Lrp4 is involved in hyperphosphine-induced VSMC calcification.GBE can reduce the high phosphorus induced VSMC calcification by regulating the Wnt/β-catenin signaling pathway.
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Objective To discover a novel long noncoding RNA YLL066W-B (referred to as YLB), whose expression can be regulated by a ubiquitin ligases E3, Huwel/Tom1, and further investigate the regulatory effects of YLB on expression of multiple subtelomeric genes. Methods Yeast strains (including Tom1△, YLB-HA, HA-YLB, pYES2-HA-YLB and YLB△) were constructed according to the principle of PCR-based tagging of yeast genes. The effects of Tom1 deletion on gene expression were analyzed by real-time PCR and DNA microarray. The protein levels were detected by Western blot. We further performed quantitative real time-PCR to analyze the inlfuence of YLB on expression of multiple subtelomerical genes.Results We found that deletion of Tom1 in yeast could affect the expression of multiple genes and greatly up-regulated the expression of YLB, which is implicated in cell cycle regulation. By analyzing its nucleotide sequence(171 bp)and detecting protein expression, we speculate that the transcriptional product of YLB is a long noncoding RNA (lncRNA). Although YLB is not homologous to any protein-encoding sequences by NCBI blast, it is homologous to the upstream or downstream regions of the open reading frame of several subtelomerically-encoded genes, including those from pau family and DNA helicase Yrf family. Thus, it is possible that YLB is involved in the regulation of these subtelomerically-encoded genes. Accordingly, deletion of YLB markedly up-regulated the mRNA levels of Yrf1-4, pau4 and pau22, whereas over-expression of YLB greatly down-regulated their expression.Conclusion We have discovered the novel lncRNA YLB. The expression of YLB could be negatively regulated by Tom1, and YLB could regulate the expression of multiple subtelomeric genes.
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The ubiquitin-proteasome pathway is responsible for the degradation of most cellular proteins in eukaryotes.It regulates almost all cellular activities, including cell proliferation, differentiation, apoptosis, gene transcription, and DNA repair.The dysfunction of the ubiquitin-proteasome pathway is associated with the pathogenesis of numerous human diseases, including cancer and neurodegenerative diseases.The marketed proteasome inhibitors have been successfully used to treat multiple myeloma and mantle cell lymphoma.Furthermore, novel inhibitors against the components of the ubiquitin-proteasome pathway are under developed and exhibit promising therapeutic effects in vivo.This paper will briefly introduce the progress on the drug discovery related to the ubiquitin-proteasome pathway.
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Objective To investigate neuroprotective mechanisms of vinpocetine by observing the effects of vinpocetine injection on the expressions of peroxisome proliferators-activated receptor γ(PPARγ),nuclear factor (NF)-κB p65,cyclooxygenase-2 (COX-2) in the ischemic cortex,and infarct volume after focal cerebral ischemia-reperfusion in rats.Methods A focal cerebral ischemia-reperfusion injury model was induced by suture method.The rats were randomly divided into a normal control,a cerebral ischemiareperfusion and a vinpocetine groups.They were also divided into either a day 7 subgroup or a day 14 subgroup (n =6 in each subgroup) according to the reperfusion time.Western blot was used to detect the expression levels of PPARγand NF-κB P65 in the ischemic cortex.Triphenyl tetrazolium staining was used to detect the volume of cerebral infarction.Results Western blot showed that at day 7 and 14 after cerebral ischemia-reperfusion,expression levels of PPARκ (all P＜0.001) and NF-κB p65 (all P＜0.001) in the cerebral ischemia-reperfusion group were significantly higher than those in the sham operation group,the expression levels of PPARκ (all P ＜0.05) in the vinpocetine group were significantly higher than those in the cerebral ischemia-reperfusion group,but the expression levels of NF-κB p65 (all P ＜0.05) were significantly lower than those in the cerebral ischemia-reperfusion group.Reverse transcription polymerase chain reaction showed that COX-2 mRNA expression levels were upregulated significantly at day 7 and 14 after cerebral ischemia-reperfusion compared with the sham operation group (all P ＜ 0.001),the expression levels of COX-2 mRNA in the vinpocetine group were significantly downregulated compared with the cerebral ischemia-reperfusion group (all P＜ 0.05).The infarct volumes at day 7 (134.308± 9.954 mm3vs.185.543 ± 9.100 mm3;q=10.659,P＜0.001) and at day 14 (137.865 ± 9.094 mm3vs.183.210±4.368 mm3;q=11.166,P＜0.001) in the vinpocetine group were significantly less than those in the cerebral ischemia-reperfusion group.Conclusions Vimpocetine significantly reduces infarct vohme after focal cerebral ischemia-reperfusion,its mechanism may be associated with upreguhtion of PPARγexpression and downreguhtion of the expressions of NF-κB p65 and COX-2.