Abstract:Aim To investigate the mechanism and signal pathways of xeroderma pigmentosum group D(XPD) gene on the proliferation of human umbilical vein smooth muscle cell (HUVSMC) induced by ox-LDL. Methods HUVSMCs were transfected with the plasmids of pEGFP-N2/XPD using Lipofectamine 2000, and subsequently silent mTOR gene. MTT and EdU assay was used to detect the cell proliferation. Flow cytometry was used to examine the cell apoptosis. The expression of XPD, lectin-like oxidized low-density lipoprotein receptor 1(LOX-1), mTOR, phospho-mTOR, Bcl-2 and Bax was measured by Western blot. Results The expression of XPD and Bax protein was down-regulated in ox-LDL group (P＜0.05), while the expression of LOX-1, mTOR, Bcl-2 protein and the ratio of Bcl-2/Bax was significantly up-regulated (P＜0.05), compared with control group. Cell proliferation of ox-LDL group increased obviously (P＜0.05). After transfected with the pEGFP-N2/XPD plasmid, the expression of Bax was significantly up-regulated, while the expression of LOX-1, mTOR, Bcl-2 and the ratio of Bcl-2/Bax were significantly down-regulated (P＜0.05). Flow cytometry showed that overexpression of XPD increased the apoptosis rate of HUVSMC (P＜0.05). MTT and BdU showed that cell proliferation of pEGFP-N2/XPD group reduced compared with control group (P＜0.05). Compared with control group, the expression of LOX-1 was significantly down-regulated in siRNA mTOR group (P＜0.05). Conclusion XPD can inhibit HUVSMC proliferation and promote its apoptosis, and reduce the effect of ox-LDL promoting proliferation of HUVSMC via the mTOR/LOX-1 pathway. XPD may be the target of treatment of atherosclerosis.

Abstract:Aim To study the effects of melatonin (MT) on homocysteine (Hcy)-induced injury of coronary artery endothelial cells and clarify whether the mechanism is related to the activation of protein kinase B (Akt)/mammalian rapamycin target protein (mTOR) pathway. Methods Human coronary artery endothelial cells (HCAEC) were cultured and grouped. The control group was treated with DMEM without drugs, the Hcy group with DMEM containing 1 mmol/L Hcy, the MT group with DMEM containing 1 mmol/L Hcy and 5 μmol/L MT, and the MT+LY group with DMEM containing 1 mmol/L Hcy, 5 μmol/L MT and 10 μmol/L LY2942. Cell viability, cell apoptosis, cell cycle and the expression of p-Akt and p-mTOR were detected. Results Cell viability,Sü phase and G2/M phase ratio, the expression of Bcl-2, cyclinD1, p-Akt and p-mTOR in Hcy group were lower than those in control group, apoptotic rate, G0/G1 phase ratio, the expression levels of Bax and cleaved caspase-3 in Hcy group were higher than those in control group; cell viability,Sü phase and G2/M phase ratio, the expression levels of Bcl-2, cyclinD1, p-Akt and p-mTOR in MT group were higher than those in Hcy group, apoptotic rate, G0/G1 phase ratio, the expression levels of Bax and cleaved caspase-3 in MT group were lower than those in Hcy group; cell viability,Sü phase and G2/M phase ratio, the expression of Bcl-2, cyclinD1, p-Akt and p-mTOR in MT+LY group were lower than those in MT group, apoptotic rate, G0/G1 phase ratio, the expression levels of Bax and cleaved caspase-3 in MT+LY group were higher than those in MT group. Conclusion MT can reduce the apoptosis of HCAEC induced by Hcy, regulating Akt/mTOR pathway is one of the mechanisms responsible for MT reducing HCAEC injury.

Abstract:Aim Based on the mTOR/ULK1 autophagy signaling pathway to discuss the mechanism of gypenoside improving aorta lipid deposition of ApoE－/－ mice. Methods 30 healthy ApoE－/－ mice were randomly divided into the model control group and the gypenoside group, and the simvastatin group, 10 mice in each group. 10 C57bL/6J mice were used as the normal control group. The model control group, the gypenoside group and the simvastatin group were fed with high-fat diet for 4 weeks. The gypenoside group and simvastatin group were treated with gypenoside 2.973 g/(kg·d) and simvastatin 2.275 mg/(kg·d) by gastrogavage for 8 weeks, respectively. The normal control group and the model control group were given by gastrogavage with the normal saline of the same volume. The formation of atherosclerotic plaque of the mice was detected by HE staining, and the blood lipid level was detected by the fully automatic biochemical analyser. The expressions of ULK1, Beclin1, LC3 and p-mTOR proteins were dectected by the Western blot.Results Compared with normal control group, in the model control group, TG, TC and LDLC were significantly increased (P＜0.05), HDLC was significantly decreased (P＜0.05), large atheromatous plaques could be seen in aortic canal, ULK1, Beclin1 and LC3 were significantly decreased (P＜0.01), p-mTOR was significantly increased (P＜0.01). Compared with the model control group, in the gypenoside group and the simvastatin group, TG, TC and LDLC were significantly decreased (P＜0.05), HDLC was significantly increased (P＜0.05), atheromatous plaques in aortic canal were significantly decreased, ULK1, Beclin1 and LC3 were significantly increased (P＜0.01), p-mTOR was significantly decreased (P＜0.01 or P＜0.05). Conclusion Gypenosides could relieve the formation of atherosclerotic plaques and prevent atherosclerosis possibly through regulating the autophagy.

Abstract:mTOR is a serine/threonine kinase, mainly involved in two kinds of signal pathway in regulation. mTOR signaling pathway can regulate cell growth, proliferation and apoptosis. While the mTOR can regulate the process of atherosclerosic development about endothelial cells migration and proliferation, macrophage autophagy, smooth muscle cells proliferation and migration. By inhibiting or activating mTOR in different periods, the vulnerable plaque of atherosclerosis can be stabilized and the development of atherosclerosis can be prevented. mTOR signaling pathway plays a multifaceted effect in atherosclerosis progression. This paper mainly review the relationship of mTOR signaling pathway and atherosclerosis. It provides a new direction for the clinical treatment.

Abstract:Aim To investigate the role of selective inhibition of the PI3K/Akt/mTOR signaling pathway on prompting rabbit primary macrophage autophagy. Methods Primary macrophages were obtained intraperitoneally from the New Zealand rabbits and then were co-cultured with the phosphatidylinositol 3-kinase （PI3K） inhibitor LY294002 （10 μmol/L）, protein kinase B （Akt） inhibitor triciribine （20 μmol/L）, mammalian target of rapamycin （mTOR） inhibitor rapamycin （10 nmol/L） and no drugs respectively for 12 hours. Ultrastructural changes of macrophages were examined by transmission electron microscopy. The expression level of microtubule-associated protein light chain 3Ⅱ（LC3Ⅱ） was assayed by immunofluorescence. Protein expression levels of Akt, mTOR, phosphorylation of protein kinase B （p-Akt）， phosphorylation of mammalian target of rapamycin （p-mTOR） and autophage related protein Beclin-1 and autophagy protein 5 and 12 conjugated form （Atg5-Atg12） were measured by Western blot. Mondansylcadaverine （MDC） staining was used to see autophagy lysosome changes. Results Compared with the control group, few autophagosomes and vacuoles were detected in group LY294002 while plenty of typical autophagosomes were detected in group rapamcin and triciribine. The expression levels of Beclin-1 and Atg5-Atg12 decreased significantly in group rapamcin, while increased significantly in group rapamcin and triciribine. The fluorescence microscope showed few dots of LC3Ⅱ in group LY294002 and many in group rapamcin and triciribine. The expression of p-Akt and p-mTOR increased obviously in group rapamcin while the former increased a lot and the latter decreased in group rapacin after co-culturing of 4 hours. The expression of p-mTOR decreased significantly in the treated groups, however, p-Akt decreased significantly in group rapamcin and triciribine but increased obviously in group rapamci after 12 hours’ co-culture. There were no significant differences on the total AKT and mTOR levels among the treated groups. MDC staining showed decreased autophagic lysosomes in group LY294002 and increased autophagic lysosomes in group rapamcin and triciribine. Conclusion Selective inhibition of PI3K/Akt/mTOR signaling pathway can promote rabbit primary macrophage autophagy while inhibition of PI3K suppress macrophage autophagy by other signaling pathway.

Abstract:Aim To investigate the plausible relationship between the polymorphism of methylenetetrahydrofolate reductase (MTHFR) gene at C677T locus and serum lipid levels and longevity in the long-lived cohort residing in Guangxi Hongshuihe River Basin. Methods Genotyping was performed with PCR-RFLP technique for 505 long-lived Zhuang individuals inhabiting in Guangxi Hongshuihe River Basin whose ages were 90 and above (long-lived group, LG) and 468 ethnic- and geographic-matched healthy mid-aged or elderly controls (non-long-lived group, non-LG). Association analysis were undertaken between MTHFR C677T genotypes and serum lipids (total cholesterol, TC triglyceride, TG high density lipoprotein, HDL low density lipoprotein, LDL). Results The allelic (C and T) and genotypic (CC, CT, and TT) frequencies of the LG and non-LG were 80.2%, 19.8% versus 85.0%, 15.0% and 65.5%, 29.3%, 5.2% versus 73.9%, 22.2%, 3.8%, respectively, which displayed significant differences between the two tested groups, with an overrepresentation of T allele in long-lived females specially, but not in males. On lipid profiles, the levels of TC, TG, LDL in LG are significantly higher than that in the non-LG. After stratifying by MTHFR C677T genotype and gender, the TC, TG, and LDL levels were noted dramatically higher in females but not in males harboring the mutant genotype (CT/TT) than that of the non-T carriers (CC) in the LG. Conclusions Our data suggest that there was a femalespecific association between the MTHFR C677T polymorphism and serum lipid levels and human longevity, which may be one of the molecular genetic basis for the longevity in the Guangxi Hongshuihe River Basin.

Abstract:Aim To evaluate the effect of LDL and ox LDL on the activity and mRNA expression of hepatic lipase (HL) in HepG2 cells. Methods Gradient concentrations (1.7～1 758 mg/L) of LDL or ox LDL was added into the medium and coincubated with HepG2 cell. The cytostasis rate of HepG2 cell was estimated by MTT and the activity and mRNA expression of HL in these cells were measured with cytochemistry and RT PCR respectively. Results In the 27.47 mg/L and 54.95 mg/L LDL groups, the mRNA expression of HL was up regulating 2.8 times and 2.3 times respectively to the cell control. In the 109.8 mg/L LDL, the mRNA expression of HL decreased 20% to the cell control. The HL activity in HepG2 cells incubated with LDL was negative related with the LDL concentration (r=-0.95614, p<0.05). The cytostasis rate in LDL increased by dose dependent model (r=0.91199, p<0.05). In the ox LDL group, the mRNA expression of HL was inhibited at all concentrations. In 27.47 mg/L and 54.95 mg/L ox LDL, the activity of HL was 3～4 times as much as that of LDL groups. Moreover, at the same concentration of 54.95 mg/L, the cytostasis rate in the ox LDL group was one time higher than in the LDL group. Conclusions At the appropriate range of concentration, LDL can induce the up regulation of mRNA expression of HL. High concentration of LDL and all concentration of ox LDL can inhibit not only mRNA expression but the activity of HL in HepG2 cells.

Abstract:Mitochondrial DNA differs from chromosomal DNA by its endosymbiotic characteristics of being a foreigner to protoeukaryotic cells circular intronless and multitronics. When mtDNA is recognized as a transgene humankind becomes a trans-genie species occurred naturally under strict definition of transgenic technology. With transgenic human assay we are able to study the biological effect of mtDNA-original homologues in the genome of modem eukaryotic cells. This new discipline is termed Mitochromics. Mitochromic analysis allows the assembling of an ancient mtDNA using eight nuclear mtDNA analogues which has an overall consensus rate of 94% with NC-001806. We believe that this assembled ancient mtDNA sequence will have enormous application in evolution and biomedical studies.

Abstract:With mimicking the injury model from murine peritoneal macrophages attacked by oxidized low density lipoprotein (OLDL), the time-de pendant and concentration-depandant effect of polysac charide K (PSK) on the foam-like change and attenuation of murine peritoneal macrophages attacked by OLDL was observed.After incubated with 10,50 and 100 mg·L-1 OLDL for 24,48,72 hOurs in vitro, the fingure of PSK-treated macrophages was maintained normal relatively.Moreover,in PSK-treated group, the amount of survival cells were higher with counted by MTT colorimetric analysis at every time points.So PSK could inhibit the foam-like change of macrophages efficiently.And after the change in macrophages formed,PSK could prolong the survival time of these macrophages.