Effect of homocysteine on the expression of macrophage inflammatory protein-1α in THP-1 monocytes

AIM: To investigate whether homocysteine (HCY) induce the expression of macrophage inflammatory protein-1α(MIP-1α)in cultured THP-1 monocytes. METHODS: After exposure of THP-1 monocytes to HCY at increasing concentrations (0.05,0.1 and 0.2 mmol/L) for 8 h, or at 0.1 mmol/L of HCY for different incubation times (4, 8 and 16 h), the expressions of MIP-1α mRNA and protein were determined by RT-PCR and immunocytochemistry, respectively. RESULTS: RT-PCR showed that the expression of MIP-1α mRNA increased with the concentrations of HCY compared with the control group. Meanwhile, after the treatment of 0.1 mmol/L HCY to the cells for different times, the MIP-1α mRNA expression increased at 4 h, peaked at 8 h, and then decreased at 16 h. The authenticity of RT-PCR products was confirmed by DNA sequencing. Image analysis of Immunocytochemistry assay showed the expression of MIP-1α protein in experimental groups increased in a dose- and time-dependent manner(P＜0.01). CONCLUSIONS: HCY induced monocytes to express MIP-1α mRNA and protein.     

Effect of homocysteine on secretion and expression of interleukin-6 in cultured rat vascular smooth muscle cells

AIM: To investigate the effect of homocysteine(Hcy) on secretion and expression of interleukin-6(IL-6), which is a multifunctional proinflammatory cytokine, in cultured rat vascular smooth muscle cells(VSMCs). METHODS: Rat VSMCs were stimulated with Hcy. Cell ELISA was performed to measure the expression of IL-6 protein and semiquantitative RT-PCR was used to dectect the IL-6 mRNA expression. RESULTS: Compared with control, treatment of 0.25 mmol Hcy for 6 h could increase IL-6 production. In addition, Hcy concentration-dependently increased the expression of IL-6 protein in these cells. 0.1 mmol/L, 0.25 mmol/L Hcy increased IL-6 production 1 4-fold and 3 4-fold, respectively Furthermore, RT-PCR analysis demonstrated that homocysteine also enhanced IL-6 mRNA expression in a concentration- and time-dependent manner.CONCLUSION: Homocysteine can induce IL-6 expression in VSMCs and elicit vascular inflammatory response, which may thereby influence the pathogenesis of atherosclerosis.     

Effect of p38 protein kinase on the activation of rat alveolar macrophages by lipopolysaccharide

AIM:To investigate the role of p38 protein kinase in the activation of rat alveolar macrophages(AMs) induced by lipopolysaccharide(LPS).METHODS:Nuclear protein was extracted, p38 protein kinase in nuclear extracts was analyzed by Western blot. The concentrations of TNF-α and IL-8 in supernatant were measured by radioimmunoassay.RESULTS:The concentrations of TNF-α, IL-8 in supernatant and p38 protein kinase in nuclear extracts were increased significantly induced by LPS and blocked by SB203580, a selective inhibitor of p38 protein kinase.CONCLUSION:The inductoin of TNF-α and IL-8 in alveolar macrophages by LPS may be mediated through the activation of p38 protein kinase.     

Effect of KLF6 gene on apoptosis of THP-1 cell-derived macrophages

AIM: To investigate the effects of Kruppel-like factor 6 (KLF6) over-expression on the viability, apoptosis, reactive oxygen species (ROS) level and AKT signaling pathway of THP-1 cell-derived macrophages. METHODS: Human monocyte cell line THP-1 was induced to differentiate into macrophages by phorbol myristate acetate (PMA), and the macrophages were randomly divided into pcDNA3.1 group, oxidized low-density lipoprotein (ox-LDL) group, ox-LDL+pcDNA3.1 group and ox-LDL+pcDNA3.1-KLF6 group. pcDNA3.1 was transfected according to Lipofectamine^TM 2000 Kit. The cell viability, apoptotic rate and ROS level were detected by MTT assay, flow cytometry with Annexin V-FITC/PI double staining and H₂DCF-DA probing, respectively. The protein levels of Bcl-2, Bax and p-AKT were determined by Western blot. RESULTS: After pcDNA3.1-KLF6 was transfected into the macrophages, the expression of KLF6 was increased significantly (P<0.05). ox-LDL significantly inhibited the viability of the macrophages, induced apoptosis and ROS production, up-regulated the protein expression of Bax, and down-regulated the protein levels of Bcl-2 and p-AKT (P<0.05). Over-expression of KLF6 significantly reduced the effects of ox-LDL on cell viability, apoptosis, ROS level and the protein levels of Bcl-2, Bax and p-AKT (P<0.05). CONCLUSION: KLF6 significantly reduces the apoptosis of THP-1 cell-derived macrophages induced by ox-LDL, which may be related to the reduction of ROS level and activation of AKT signaling pathway.     

Effect of homocysteine on the apoptosis of cultured human umbilical vein endothelial cells

AIM: To investigate the effect of homocysteine(Hcy) on the apoptosis of endothelial cells (EC). METHODS: First-passaged human umbilical vein endothelial cells (hUVEC) were cultured with M199 containing 3 mmol/L Hcy. hUVEC apoptosis was detected as follow: demonstration of nuclear changes by Hoechst 33258 staining, agarose gel electrophoresis of DNA fragments, detection of apoptotic cells by flow cytometry following Annexin V-PI doubled stain, Western blot for P53 and Bax protein detection and colorimetry detecting caspase-3 activity. RESULTS: Compared with control, homocysteine induced characteristic apoptotic changes in hUVEC. The chromosomal DNA of hUVEC appeared "DNA ladder" by agarose gel electrophoresis. Apoptotic cells were increased significantly (P<0.01, n=3). Hcy promoted the expression of protein Bax, P53 (P<0.01, n=3) and enhanced the activity of caspase 3 (P<0.05, n=3). CONCLUSION: Homocysteine induces apoptosis in cultured hUVEC.     

Regulation of ATP-binding acssette transporter A1 on apolipoprotein E secretion from macrophages

AIM: To investigate the regulatory effect of the adenosine triphosphate binding cassette transporter A1 (ABCA1) on apolipoprotein E secretion from human THP1 macrophages.METHODS: Differentiation of THP1 macrophages from monocytes was stimulated with phorbol 12-myristate 13-acetate. The macrophages then were incubated with factors which regulate ABCA1 expression. After periods of incubation, apo E secreted in the medium and synthesized in the cell was determined with ELISA, and apo E mRNA espression was detected with Northern blot.RESULTS: An increase in apo E secretion from THP1 macrophages was observed by 8 h of incubation with 8-Br-cAMP, an activator of ABCA1 expression (P＜0.05). Glyburide, a putative ABCA1 inhibitor, and antisense oligonucleotides specifically against ABCA1 mRNA remarkably decreased apo E secretion from both THP1 macrophages and macrophage foam cells (P＜0.01,respectively). Neither apo E mRNA expression nor intracellular apo E level in THP1 macrophages was changed by inhibition of ABCA1.CONCLUSION: ABCA1 may promote the secretion of apo E from macrophages and macrophage foam cells and the effect may occur at the level of post-translation. The present results reveal a new aspect underlying antiatherogenic properties of ABCA1.     

Influence of Salvia miltiorrhiza injection on lipid and ICAM-1 expression in rats with atherosclerosis

AIM: To study the effect of Salvia miltiorrhiza injection on rat atherosclerosis (AS), and elucidate the possible mechanism. METHODS: Wistar rats were fed with fat-rich diet and high dose of vitamin D₃ to induce AS, then treated with Salvia miltiorrhiza injection. Concentrations of triglyceride (TG) and total cholesterol (TC) in serum were measured by automatic serum biochemical assay. The level of ICAM-1 protein and mRNA were determined by Western blot and RT-PCR. RESULTS: Compared with the AS model group, the levels of TG and TC in serum were significantly lower in Salvia miltiorrhiza injection group (P<0.05). Western blot and RT-PCR showed that the level of ICAM-1 protein and mRNA were decreased in Salvia miltiorrhiza injection group compared with AS group. CONCLUSION: Salvia miltiorrhiza injection decreased blood lipid and reduced the ICAM-1 gene expression in rats with atherosclerosis.     

Effect of YIGU capsule on IGF-I mRNA and protein expression in rat osteoblasts in vitro

AIM: The effects of YIGU capsule on proliferation and IGF-I mRNA protein expressions in osteoblasts were studied. METHODS: (1) Forty 12-month old Sprague-Dawley female rats were divided randomly into four groups (YIGU capsule high dose group, medium dose group and low dose group; saline group), the drug-containing serum and control serum were prepared. (2) The new-born Sprague-Dawley rat osteoblasts were cultured with different YIGU capsule drug-containing serum at different concentrations and different exposure time. MTT method was used to observe proliferation of osteoblasts. (3) RT-PCR method was used to measure the relative IGF-I mRNA levels and ELISA method was used to measure IGF-I secretion at different exposure time. (4) ELISA method was used to measure IGF-I secretion at different exposure time. RESULTS: (1) Proliferation of osteoblasts was more than the control groups after 48, 72 and 96 h, respectively (P<0.01); (2) The relative IGF-I mRNA levels and IGF-I protein expression were higher than those in control group after 48, 72 and 96 h, respectively (P<0.01 or P<0.05). CONCLUSIONS: It was suggested that YIGU capsule drug-containing serum promoted proliferation, IGF-I mRNA and protein expression. These results may be parts of the mechanisms of YIGU capsule to prevent and treat osteoporosis.     

Taurine reduced oxygen free radical production induced by homocysteine in electron transport chain and homocysteine inhibites taurine transporter in mitochondria membrane

AIM: To observe effects of homocysteine and antagonized effects of taurine on electronic leakage and free radical production in myocardial mitochondria. METHODS: Myocardial mitochondria of rat heart was isolated, and was broken by supersonic wave to prepare submitochondria. Recombinant of succinic acid cytochrome c reductase was prepared with mitochondria of porcine heart. They were co-incubated with homocysteine and/or taurine with various concentration. The H₂O₂ and O₂^- were determined by chemiluminescence methods. The taurine transporter of heart mitochondria and its propert, and effects of homocysteine on its function were studied with glass filter. RESULTS: Homocysteine stimulated oxygen free radical production in heart mitochondria, submitochondria, and succinic acid cytochrome c in a concentration-dependent manner. Although taurine itself did not affect oxygen free radical production, taurine did inhibit oxygen free radical production in mitochondria, submitochondria and succinic acid cytochrome c in a concentration-dependent manner. Taurine transporters of Na⁺-dependent were existed in mitochondria membrane. Homocysteine inhibited taurine transtport in mitochondria in a concentration-dependent manner. CONCLUSIONS: Taurine inhibited electronic leakage and oxygen free radical production induced by homocysteine in electron transport chain. There were taurine transporters in mitochondria membrane, and transport functions of taurine transporter were inhibited by homocysteine.     

Adipophilin accumulates cellular cholesteryl ester by PKC and ACAT1 in THP-1 macrophages

AIM: To investigate the mechanism of adipophilin accumulating cellular cholesteryl ester in THP-1 macrophages. METHODS: New Zealand rabbit atherosclerotic model was made with high cholesterol diet for 12 weeks. The expressions of adipophilin and PKCα were determined by Western blotting and immunochemical staining in aortic arteries. Cholesteryl ester-loading cells (CE-loading cells) were made from THP-1 macrophages incubated with oxidized low density lipoprotein. In CE-loading cells, expressions of adipophilin and ACAT1 were analyzed by RT-PCR, and the activity of PKC was determined by PepTag assay and spectrophotometry. When the CE-loading cells were incubated with PKC activator PMA and inhibitor calphostin C, expression of adipophilin was observed with RT-PCR, and cellular lipid was measured with oil red O staining and HPLC. The pcDNA3.1-HA-adi vector was transfected to THP-1 macrophage for making adipophilin over expression cells. After the CE-loading adipophilin over expression cells were incubated with or without ACAT inhibitor, the ACAT1 expression and cellular cholesteryl ester were analyzed. RESULTS: Compared with control, both adipophilin and PKCα expression increased in aortic arteries of atherosclerotic animal. In CE-loading THP-1 macrophages, adipophilin and ACAT1 highly were expressed and PKC activity was augmented also. PMA enhanced the high expression of adipophilin and cellular cholesteryl ester in CE-loading THP-1 macrophages, but calphostin C inhibited the effect. ACAT1 expression and cellular cholesteryl ester increased in adipophilin over expression cells, the effect was impaired by incubating with ACAT inhibitor. CONCLUSION: The results suggest that adipophilin increases ACAT1 activity through enhancing PKC activity, resulting in cellular cholesteryl ester accumulation in THP-1 macrophages.     

Effect of erigeron on intercellular adhesion molecule-1 and its mRNA expression during cerebral ischemia and reperfusion in rats

AIM: To investigate the effect of erigeron on intercellular adhesion molecule-1 (ICAM-1) and mRNA expression during cerebral ischemia/reperfusion. METHODS: The rat models of middle cerebral artery (MCA) focal cerebral ischemic reperfusion were established with the suture method in the study. The ICAM-1 mRNA and protein expression were measured by RT-PCR and immunohistochemistry techniques, respectively. RESULTS: By down-regulating the expression of ICAM-1 protein and mRNA and alleviating inflammation in cerebral ischemic region, erigeron exerted a protective effect in cerebral ischemia and reperfusion. CONCLUSION: The results suggest that erigeron protects the brain against cerebral ischemia and reperfusion injury via inhibiting ICAM-1 expressino.