TNF-α exacerbates cholesterol accumulation via down-regulating LXRα promoter activity in HepG2 cells

AIM: To investigate the exacerbating effect of tumor necrosis factor alpha (TNF-α) on lipid accumulation in HepG2 cells by inhibiting liver X receptor alpha (LXRα) signaling pathway. METHODS: Luciferase reporter plasmid driven by the LXRα promoter (pGL3-Basic-LXRα-P) was constructed and transfected into HepG2 cells to detect the LXRα promoter activity. HepG2 cells were incubated with serum-free medium (control), 20 μg/L TNF-α (TNF-α), 100 mg/L LDL (LDL) and 20 μg/L TNF-α plus 100 mg/L LDL (LDL+TNF-α), respectively. The effects of TNF-α on cholesterol accumulation were examined by oil red O staining and quantitative intracellular cholesterol assay. The expression of LXRα, ABCA1 and ABCG1 at mRNA and protein levels was examined by real-time PCR and Western blotting. RESULTS: The pGL3-Basic-LXRα-P was constructed successfully. TNF-α decreased the activity of LXRα promoter in the absence or presence of LDL. Inflammatory stress inhibited the expression of LXRα, ABCA1and ABCG1 at mRNA and protein levels. The cholesterol efflux was increased after loading of LDL, while TNF-α decreased intracellular cholesterol efflux. The results of oil red O staining and quantitative intracellular cholesterol assay demonstrated that inflammatory stress increased cholesterol levels in HepG2 cells. CONCLUSION: TNF-α exacerbates the cholesterol accumulation in hepatic cells via inhibiting LXRα promoter activity and gene expression.     

Relationship between HDL subclasses and TC/HDL-C,TG/HDL-C ratio

AIM: To study the relationship between HDL subclasses and TC/HDL-C, TG/HDL-C ratio in serum. METHODS: Apolipoprotein (apo) A-Ⅰ contents of serum HDL subclasses in 292 subjects were determined by two-dimensional gel electrophoresis associated with immunodection method. RESULTS: Compared with low TC/HDL-C ratio subgroup, apoA-Ⅰcontents of preβ1-HDL (P<0.01, in middle or high subgroup) and HDL3a (P<0.05, in high subgroup) were significantly higher, but those of HDL2b (P<0.01, in middle or high subgroup) and HDL2a (P<0.01, in high subgroup) were significantly lower. Compared with middle TC/HDL-C ratio subgroup, apoA-Ⅰ contents of preβ1-HDL, HDL3a were significantly higher (P<0.01, P<0.05), but those of HDL2a and HDL2b were significantly lower (P<0.01, P<0.01) in high subgroup. Compared with low TG/HDL-C ratio subgroup, apoA-Ⅰ contents of preβ1-HDL, HDL3a were significantly higher (P<0.01, P<0.05, in high subgroup), but those of HDL2a and HDL2b were significantly lower (P<0.01, in middle or high subgroup). Compared with middle TG/HDL-C ratio subgroup, apoA-Ⅰ contents of preβ1-HDL, HDL3a were significantly higher (P<0.01, P<0.05), but those of HDL2a and HDL2b were significantly lower (P<0.01). In addition, TC, TG, TC/HDL-C ratio and TG/HDL-C ratio showed a positive correlation with apoA-Ⅰ contents of small-sized preβ1-HDL and a negative correlation with those of large-sized HDL2b, but it was reversed for HDL-C. CONCLUSION: When TC/HDL-C>5 or TG/HDL-C>2.2 in serum, the particle size of HDL shifted towards smaller sizes, which indicates that reverse cholesterol transport might be weakened and HDL maturation might be abnormal.     

Influences of apolipoprotein CII concentrations on high-density lipoprotein subclass distribution

AIM: To investigate the influence of serum apolipoprotein (apo) CII concentrations on the distribution of serum high-density lipoprotein (HDL) subclasses. METHODS: Serum HDL subclasses in 247 subjects were determined by two dimensional gel electrophoresis-immunodetection. RESULTS: With the increase in serum apolipoprotein CII levels, age, BMI, the contents of TG, TC, apoB100, apoCII, apoCIII, apoE, preβ1-HDL, preβ2-HDL, HDL3b and HDL3a increased significantly, but the contents of HDL-C, HDL2a and HDL2b decreased remarkably. The contents of preβ1-HDL increased with the rise in apoCII and apoA I levels, whereas the content of HDL2b increased with the rise in serum apoA I level in the same apoC II group, but decreased with the increase in serum apolipoprotein CII level in the same apoA I group. With the increase in the ratio of apoCII/ apoCIII, the content of preβ1-HDL elevated, but the content of HDL2b decreased. The correlation analysis illustrated that the apoCII level was positively correlated with preβ1-HDL (r=0.186, P<0.01), but inversely correlated with HDL2b (r=-0.149, P<0.05). The apoA I level was positively associated with all HDL subclasses (r in the range of 0.349-0.587, P<0.01). In addition, the apoCIII level was positively correlated with preβ1-HDL (r=0.184, P<0.01) and preβ2-HDL (r=0.178, P<0.01), while the apoE level was positively correlated with HDL3a (r=0.040, P<0.05). The apoB100 level was inversely correlated with HDL2a (r=-0.102, P<0.05). CONCLUSION: The particles of HDL show a general shift towards smaller size with the increase in apoCII levels, indicating that the maturation of HDL is abnormal. Whereas the contents of apoA I level correct the effect of apoCII on the distribution profile of HDL subclasses. The ratio of apoCII/apoCIII might also been taken as one of the indexes reflecting the distribution profile of serum HDL subclasses.     

Effect of promoting cellular cholesterol efflux on the apoptosis of foam cells derived from monocytes

AIM: To determine the effect of promoting cellular cholesterol efflux on the apoptosis of foam cells derived from monocytes. METHODS: RAW264.7 cells were incubated with 50 mg/L ox-LDL as a foam cell mode. The apoptosis rate of RAW264.7 cells was assayed by flow cytometry. Cellular lipid droplet was assayed by oil red staining. The rate of cellular cholesterol efflux was assayed with ［3H］ label cholesterol, and the content of cellular cholesterol were assayed by high performance liquid chromatography. RESULTS: After incubation with 50 mg/L ox-LDL for 48 h, the content of cellular cholesterol ester increased from (6.8±3.6) mg/g to (101.7±4.5) mg/g (P<0.05), but the rate of cholesterol efflux was only (5.2±2.1)%, and the apoptosis rate of RAW264.7 was increased from 8.14% to 42.6% (P<0.05). When treated with 200 mg/L HDL3 for 24 h, the rate of cellular cholesterol efflux were obviously increased, the apoptosis rate were decreased from 42.6% to 14.3% (P<0.05). Meanwhile, when treated with 10 mmol/L β-CD (a special compound for promoting cellular cholesterol efflux) for 24 h, the apoptosis rate was also decreased from 42.6% to 12.0%. CONCLUSION: HDL3 and β-CD inhibit the apoptosis of foam cells induced by oxidized LDL through promoting cellular cholesterol efflux.     

The interrelationships between serum HDL subpopulations and triacyglycerol

AIM: To Study the influence of plasma TG level on the contents of serum HDL subpopulations. METHODS: Classified by the contents of serum TG, the serum level of HDL subpopulations in 106 normal TC and 183 high TC subjects were analyzed by two-dimensional gel electrophoresis coupled with immunodection method. RESULTS: The apo-AⅠcontents of per-β1-HDL, HDL3c, HDL3b and HDL3a were higher in a certain extent in TC high subjects vs corresponding TC desirable subjects. The apo-AⅠ contents of per-β1-HDL （in borer-line high TG subgroup） and HDL3b （in very high TG subgroup） were significantly higher (P<0.05 or P<0.01), while the apo-AⅠ contents of HDL2b and HDL2a were all lower in TC high subjects vs corresponding TC normal subjects. With the increase in plasma TG levels, the apoA-Ⅰ content of pre-β1-HDL increased, and it was significantly higher (P<0.05 or P<0.01) in borderline-high TG(except TC desirable subjects), high TG and very high TG subgroups vs corresponding normal TG subgroup. Contents of HDL3b and HDL3a had the same tendency, and in TC high subjects contents of HDL3b (in very high TG subgroup) and HDL3a (in every subgroups, in which levels of TG were higher) were significantly higher (P＜0.05 and P＜0.01） than in normal TG subgroup. On contrast, the apoA-Ⅰcontents of HDL2b and HDL2a following with the increase of plasma TG levels tended to become lower. Contents of HDL2b were significantly lower (P<0.05) in high TG subgroup and very high TG subgroup vs corresponding normal TG subgroup, and in TC high subjects contents of HDL2a were significantly lower (P<0.05) in very high TG subgroups vs normal TG subgroup. CONCLUSION: Our data show the general shift toward smaller size of HDL particle size with the increase in TC and TG levels. Contents of TC and TG are very important to contents of HDL subclasses.     

Relationship between subclasses of serum HDL and LPL gene HindⅢ polymorphism in hyperlipidemia

AIM: To investigate lipoprotein lipase gene HindⅢ polymorphism and its relationship with serum lipids and apolipoprotein, serum HDL subclasses in patients with hyperlipoidemia. METHODS: Lipoprotein lipase gene HindⅢ polymorphism was assayed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The subclasses of serum HDL in 152 hyperlipoidemia patients and 128 healthy subjects were determined by two-dimensional gel electrophoresis conjunction with immunodetection method. RESULTS: H+H+ genotype and allele H+ in hyperlipoidemia and control groups were both the highest. In hyperlipidemia group, H+H+ genotypes tended to be higher than that in control group, while H+H- and H-H- genotypes were significantly lower (P<0.05). In hyperlipidemia group allele H+ carriers' frequency tended to be higher than that in control group (P<0.05). In hyperlipoidemia group, the genotype of H+H+ showed higher serum TG, apoB100 levels, TG/HDL-C ratio, preβ1-HDL, HDL3b and lower HDL2a, HDL2b compared with H-H- (P<0.05). In control group, the genotype of H+H+ had higher serum TG，HDL3c and lower HDL2a compared with H-H- (P<0.05). CONCLUSION: The HindⅢ polymorphism at intron 8 of LPL gene is associated with the general shift toward smaller size of HDL particle size in hyperlipoidemia, and the change of HDL subclasses distribution profile may be closely related to the pathogenesis of atherosclerosis in Chinese patients with hyperlipoidemia.     

Inhibitory effect of Wendan decoction on formation of foam cells induced by ox-LDL

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on formation of foam cells. METHODS The optimal concentrations of Wendan decoction without cytotoxity to cells were selected by MTT assay. After Wendan decoction treatment, the formation of foam cells was examined by oil red O staining. The cholesterol efflux, cholesterol level, free cholesterol level and cholesterol esterification rate were analyzed using cholesterol efflux assay, total cholesterol assay and free cholesterol assay. The expression levels of macrophage membrane proteins, including CD36, scavenger receptor class A （SR-A）, ATP-binding cassette transporter A1 （ABCA1） and scavenger receptor class B type I （SR-BI）, were quantified by Western blot. RESULTS The optimal concentrations of Wendan decoction without cytotoxity to the cells were 0~6 g/L. Wendan decoction at the concentrations of 1.5, 3 and 6 g/L were selected for the experiments. Wendan decoction at these concentrations inhibited the formation of foam cells induced by oxidized low-density lipoprotein （ox-LDL）, and reduced the accumulation of intracellular lipids in a concentration-dependent manner （P<0.05 or P<0.01）. Wendan decoction also reduced intracellular total cholesterol level, cholesterol ester level and cholesterol esterification rate （P<0.05 or P<0.01）, promoted efflux of intracellular cholesterol （P<0.01）, and decreased the protein level of CD36 in THP-1 cell-derived macrophages （P<0.01） in a concentration-dependent manner. Wendan decoction at the concentration of 6 g/L significantly reduced the protein level of SR-A in THP-1 cell-derived macrophages （P<0.05）. At the concentrations of 3 and 6 g/L, Wendan decoction significantly increased the protein levels of ABCA1 and SR-BI in THP-1 cell-derived macrophages （P<0.05 or P<0.01）. CONCLUSION Wendan decoction significantly inhibits ox-LDL-induced formation of foam cells by reducing cholesterol deposition and promoting cholesterol efflux, and its mechanism may be related to the down-regulation of CD36 and SR-A and the up-regulation of ABCA1 and SR-BI.     

Action of ATP binding cassette transporter A 1 on cholesterol efflux in THP-1 macrophage-derived foam cells

AIM:To study the action of ATP binding cassette transporter(ABC) A 1 on cholesterol efflux in THP-1 macrophage-derived foam cells.METHODS:After exposure of the cultured THP-1 macrophage-derived foam cells to 22(R)-hydroxycholesterol and 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) at different concentration for 24 hours, cholesterol efflux and ABCA1 mRNA level were determined by FJ-2107P type liquid scintillator and reverse trancriptase-polymerase chaim reaction(RT-PCR), respectively.RESULTS:Oxidized LDL promoted cholesterol efflux in THP-1 macrophages and 22(R)-hydroxycholesterol increased cholesterol efflux in THP-1 macrophage-derived foam cells in a dose-dependent manner and DIDS inhibited cholesterol efflux in THP-1 macrophage-derived foam cells in a dose-dependent manner. Exposure of the cultured THP-1 macrophage-derived foam cells to 22(R)-hydroxycholesterol and DIDS at different concentration for 24 hours, resulted in increase and decrease in the expression of ABCA1 mRNA in THP-1 macrophage-derived foam cells in a dose-dependent manner, respectively.CONCLUSION:ABCA1 playes an important role in cholesterol efflux in THP-1 macrophage-derived foam cells.     

Effect of puerarin on cholesterol influx and efflux in RAW264.7-treated foam cells

AIM: To study the protective effect of puerarin on the atherosclerosis of RAW264.7-derived foam cells. METHODS: The model of foam cells was established by incubating the RAW264.7 cells with ox-LDL. The cholesterol uptake was evaluated by a DiI-ox-LDL binding assay. The ability of cholesterol efflux of the RAW264.7-derived foam cells was detected by cholesterol efflux assay. The protein levels of LC3Ⅱ, P62, CD36, ABCA1, LAL and p-AMPK were determined by Western blot. RESULTS: Puerarin treatment reduced the cholesterol uptake capacity and enhanced the cholesterol efflux rate. The protein levels of LC3Ⅱ, ABCA1 and LAL in puerarin group were higher than that in ox-LDL group, while the protein levels of P62 and CD36 were obviously decreased, and those in rapamycin treatment group had the same change as puerarin group. The protein levels of LC3Ⅱ, ABCA1 and LAL were obviously decreased and the protein level of p-AMPK was increased after co-treated with 3-MA. CONCLUSION: Puerarin promotes LAL and ABCA1-mediated cholesterol efflux in ox-LDL-treated RAW264.7 macrophages, which might enhance autophagy through AMPK-dependent pathway for cholesterol efflux regulation, and reduce the uptake of lipids by CD36 negative regulation.     

Role of apolipoprotein E in cholesterol efflux mediated by ABCA1 and ABCG1

AIM:To investigate the role of apolipoprotein E(ApoE) in cholesterol efflux mediated by ATP-binding cassette transporter A1(ABCA1) and ATP-binding cassette transporter G1(ABCG1). METHODS:RAW 264.7 cells were seeded in either 6-well or 24-well plates, and then incubated with 20 mg/L low-density lipoprotein receptor gene knockout(LDLr^-/-) mouse lipoprotein 20 mg/L ApoE gene knockout(ApoE^-/-) mouse lipoprotein or culture medium alone. The changes of intracellular lipid content were measured by transmission electron microscopy and enzymatic colorimetric method. The cholesterol efflux was determined by liquid scintillation. The mRNA and protein levels of ABCA1 and ABCG1 were detected by real-time PCR and Western blotting, respectively. RESULTS:The ApoE^-/- mouse lipoprotein increased the content of intracellular cholesterol ester by 60% compared with the control cells. In addition, ApoE^-/- mouse lipoprotein treatment decreased the cholesterol efflux to apolipoprotein A-I(ApoA-I) and high-density lipoprotein(HDL) compared with LDLr^-/- mouse lipoprotein treatment. ApoE^-/- mouse lipoprotein treatment inhibited the mRNA and protein levels of ABCA1 and ABCG1 compared with LDLr^-/- mouse lipoprotein treatment. CONCLUSION:Apolipoprotein E plays an important role in the cholesterol efflux of macrophages, which is associated with its regulatory effect on the expression of ABCA1 and ABCG1.     

Effects of oxidized low density lipoprotein and lysophosphatidylcholine on cholesterol efflux from mouse peritoneal macrophage foam cells

AIM:To explore the effects of oxidized low density lipoprotein (OxLDL) and one of its component— lysophosphatidylcholine (LPC) on cholesterol efflux from mouse macrophage foam cells.METHODS:(1) Cholesterol efflux induced by apoAI from mouse peritoneal macrophage foam cells loaded with OxLDL or acylated LDL(AcLDL) was measured. (2) Cholesterol efflux induced by LPC and apoAI from macrophage foam cells separated from normal or apoE gene deficient (E⁰) mouse loaded with AcLDL were measured.RESULTS:(1) When the macrophage foam cells were incubated with apoAI, cholesterol efflux from AcLDL-induced macrophage foam cells increased significantly compared to that of OxLDL-induced macrophage foam cells. (2) LPC promoted cholesterol efflux from macrophage foam cells in relation to both dosage and time. When LPC was incubated with E⁰ mouse macrophage foam cells, the released cholesterol mass was significantly lower than that of normal mouse macrophage foam cells. It was also found that cholesterol efflux induced by apoAI normally occurred in E⁰ mouse macrophage foam cells.CONCLUSIONS:(1) OxLDL accumulated cholesterol in macrophages and impair cholesterol efflux. (2) LPC induced cholesterol efflux from macrophage foam cells, which may occur via apoE pathway.     

Subclasses of serum HDL in middle and old aged patients with hyperlipidemia in Chengdu City

AIM: To detect the change of composition and ratio of serum HDL subclasses and explore the relationship between these changes and the plasma lipid level in patients with hyperlipidemia. METHODS: The components of subclasses of serum HDL in 172 middle and old aged patients with hyperlipidemia and 115 healthy middle and old aged were determined by dimensional gel electrophoresis associated with immuno-blotting method. RESULTS: Compared to the healthy controls, the contents of pre β1-HDL，　HDL3b and HDL3a were significantly higher (P＜0.05 or P＜0.01), while that of HDL2b was significantly lower (P＜0.01) in middle and old aged patients with hyperlipidemia. The content of pre β1-HDL increased with age in healthy controls, whereas the HDL2b decreased. The content of pre β1-HDL was significantly higher (P＜0.05), while the HDL2b (P＜0.05) was significantly lower in men than in women in patients with hyperlipidemia and the healthy controls. In middle and old aged patients with hyperlipidemia, the content of pre β1-HDL was positively correlated with the serum TG, TC, apoB100, apoCⅡ, apoCⅢ, apoE and TG/HDL-C (r=0.432; r=0.243; r=0.341; r=0.259; r=0.335; r=0.308 and r=0.453, P＜0.05 or P＜0.01), while it was negatively correlated with HDL-C (r=－0.167, P＜0.05). The content of HDL2b was negatively correlated with TG, TC, apoCⅡ, apoCⅢ and TG/HDL-C (r=－0.296; r=－0.156; r=－0.182; r=－0.216; r=－0.203 and r=－0.313, P＜0.05 or P＜0.01), while it was positively correlated with HDL-C (r=0.124, P＜0.05). CONCLUSIONS: The particle of HDL in the middle and old aged patients with hyperlipidemia showed a general shift towards smaller size, which indicated that the reverse cholesterol transport might be weakened. Men had smaller HDL particle size than women.     

27-Hydroxycholesterol modulates lung cancer cell proliferation by activation of LXR signaling pathway

AIM:To investigate the effect of cholesterol metabolite 27-hydroxycholesterol (27-OHC) on the proliferation of lung cancer cells. METHODS:Human lung cancer A549 cells were treated with 27-OHC at different concentrations (0, 0.3125, 0.625, 1.25, 2.5, 5 and 10 μmol/L) for 24~48 h. The cell viability, cell cycle, cell prolife-ration, the intracellular cholesterol levels and cholesterol metabolism-related molecule expression were subsequently assessed by CCK-8 assay, flow cytometry, EdU staining, tissue total cholesterol detection kit, real-time PCR and Western blot. RESULTS:27-OHC decreased the viability of the A549 cells in a dose-and time-dependent manner (P<0.01) and inhibited the cell proliferation (P<0.05). The expression of typical liver X receptor (LXR) downstream target proteins including ATP-binding cassette transporter A1 (ABCA1), low-density lipoprotein receptor (LDLR), and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CR) were modulated, which promoted the efflux of intracellular cholesterol, and reduced cholesterol influx and de novo synthesis, resulting in decreased intracellular cholesterol levels and cell viability. Furthermore, the inhibitory effect of 27-OHC on A549 cell viability was significantly attenuated after the LXR pathway was partially blocked by 5 μmol/L GSK2033 treatment (P<0.05). CONCLUSION:27-OHC inhibits A549 cell prolife-ration via activation of LXR signaling pathway.     

Relationship between HDL subclass distribution and plasma glucose and lipid levels in patients with metabolic syndrome

AIM: To investigate high-density lipoprotein(HDL) subclass distribution and to analyze the relationship between HDL subclasses with plasma glucose and lipids in metabolic syndrome(MS). METHODS: Apolipoprotein A-I(apoA-I) contents of plasma HDL subclasses were determined by two-dimensional gel electrophoresis associated with immunodetection. The concentrations of lipids and apolipoproteins in the plasma were measured by an automated biochemical analyzer. RESULTS: Compared with the controls, the levels of fasting plasma glucose(FPG), total cholesterol(TC), triglyceride(TG), low-density lipoprotein cholesterol(LDL-C), LDL-C/high-density lipoprotein cholesterol(HDL-C), apolipoprotein B₁₀₀(apoB₁₀₀), apoB₁₀₀/apoA-I, systolic blood pressure(SBP), body mass index(BMI) and HDL_3b were increased in the MS patients(P<0.05). Meanwhile, HDL-C, apoA-I and preβ₂-HDL, HDL_2a and HDL_2b were decreased in the MS patients(P<0.01). With the increase in the plasma glucose level, the contents of HDL_2a and HDL_2b were decreased in the MS patients(P<0.05), while preβ₁-HDL was increased(P<0.05). With the decrease in the HDL-C level, the content of HDL_2b was decreased in the MS patients(P<0.01), while preβ₁-HDL was increased(P<0.01). With the increase in the TG level and the decrease in the HDL-C level, the content of HDL_2b had a decreasing trend and the content of small-particle preβ₁-HDL had an increasing trend, indicating that HDL maturation metabolism was disrupted. The correlation analysis showed that FPG was negatively correlated with the levels of HDL_2a and HDL_2b, HDL-C was negatively correlated with the level of preβ₁-HDL and positively correlated with the level of HDL_2b, and TG was positively correlated with the levels of preβ₁-HDL and HDL_3b. CONCLUSION: With the increases in the plasma glucose and TG, and the decrease in HDL-C in the MS patients, HDL particles have minifying tendency, and the maturation metabolism of HDL particles is disrupted.     

Relationship of subclasses of serum HDL and Apo A-Ⅰ gene polymorphism in hyperlipidemia

AIM: To investigate apolipoprotein A-Ⅰ gene (Apo A-Ⅰ) polymorphism and its relationship with serum HDL subclasses in patients with hyperlipidemia (HL). METHODS: Apo A-Ⅰ genotype was assayed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The subclasses of serum HDL in 118 patients with hyperlipidemia and 109 healthy subjects were determined by two-dimensional gel electrophoresis conjunction with immunodetection method. RESULTS: Both in HL group and the control group, G/G and C/C genotypes were the most frequent at －78 bp and ＋83 bp of Apo A-Ⅰ gene, respectively. The frequency of rare A allele at －78 bp in HL group was significantly higher than that in control group. In HL group, subjects with G/A mutation had higher serum levels of TG, Apo C-Ⅲ, pre β1-HDL and HDL3a, and lower levels of HDL2a and HDL2b compared to the subjects with G/G genotype. CONCLUSION: The G/A transition in the －78 bp position of the Apo A-Ⅰ gene promoter in patients with hyperlipidemia is associated with HDL subclasses. There is a general shift toward smaller sized HDL, which, in turn, indicates that HDL maturation might be abnormal.     

Inflammation disrupts cholesterol efflux in human kidney mesangial cells via PPARγ-LXRα-ABCA1 pathway

AIM: To investigate the perturbative effects of inflammatory stress on cholesterol efflux in human kidney mesangial cells (HMCs) and the relation to peroxisome proliferators activated receptor-γ (PPARγ)-1iver X activated receptor-α (LXRα)-and ATP-binding cassette transporter A1 (ABCA1) pathway. METHODS: HMCs were cultured and divided into control group (incubated with serum free medium), high lipid group , inflammatory stress group or combination treatment group . The mRNA and protein levels of PPARγ, LXRα,ABCA1 were examined by real-time polymerase chain reaction (PCR) and Western blotting. cholesterol assay was performed to evaluate the efflux of cholesterol by liquid scintillation counter. Oil red O staining was used to evaluate lipid droplet accumulation in the cells. Intracellular cholesterol level was measured by enzymic assay. RESULTS: : LDL increased the expression of PPARγ, LXRα and ABCA1 at mRNA and protein levels in HMCs, while TNF-α reduced the expression of these genes at mRNA and protein levels. The cholesterol efflux was increased after LDL loading. However, inflammatory stress inhibited cholesterol efflux in the absence or presence of LDL loading. Oil red O staining and quantitative analysis showed that LDL loading increased the intracellular cholesterol level in HMCs and inflammatory stress further exacerbated the lipid accumulation. CONCLUSION: Inflammatory cytokine reduces cholesterol efflux by inhibiting the expression of PPARγ, LXRα and ABCA1, thereby causing lipid accumulation in HMCs.     

Effect of proprotein convertases on transforming growth factor β1-induced HBV replication inhibition

AIM: To study the effect of proprotein convertases (PCs) on the transforming growth factor (TGF) β1-induced inhibition of HBV replication.METHODS: HepG2.2.15 cells cultured regularly were exposed to recombinant TGFβ1 at concentration of 2 μg/L or 5 μg/L and/or PC inhibitor at concentration of 20 μmol/L for 18 h. The total RNA and HBV core particle DNA were extracted from these cells, and PC mRNA and core-associated HBV DNA were detected by real-time PCR technique. RESULTS: The mRNA expression levels of 7 PCs in HepG2.2.15 cells were observed with various degrees. Recombinant TGFβ1 significantly up-regulated the mRNA expression of all PCs except for the down-regulation of PC5/6, though PC1/3 and PC2 were up-regulated most obviously. Furin and PACE4 were the predominant PCs before and after TGFβ1 exposure when the basic mRNA expression was taken into account. Further study showed that TGFβ1-induced the inhibition of HBV replication was abrogated by PC inhibitor in HepG2.2.15 cells. CONCLUSION: TGFβ1-induced the inhibition of HBV replication is mediated by the up-regulation of PCs, which might be of many implications in efficient interferences of TGFβ1 on HBV replication.