CD36/Src/ERK pathway is involved in process of monocyte differentiation into macrophages

AIM To investigate the role of fatty acid translocase (FAT/CD36) on differentiation of monocytes to macrophages. METHODS Human monocyte THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA) at 0, 100 and 200 μg /L. Small interfering RNA (siRNA) targeting CD36 (siCD36) was employed to knock down the expression of CD36 in THP-1 cells. The CD36 over-expression (CD36OE) cell line was constructed by transfection with a recombinant lentivirus containing CD36 cDNA. Optical microscopy and crystal violet staining were used to detect the monocyte morphological changes and adhesion ability. The protein expression of CD36 was measured by flow cytometry and Western blot. The mRNA levels of CD36, CD11b and CD80 were detected by real-time PCR. The protein levels of extracellular signal-regulated kinase (ERK） and Src tyrosine kinase were determined by Western blot. RESULTS The cellular adhesiveness of THP-1 cells was elevated in the process of monocytes differentiation, and the expression of CD36 was increased in this process as well (P<0.01). siCD36 was transfected into the THP-1 cells (CD36i group) and the silencing efficiency was approximately 80%. The cell surface area and cellular adhesiveness were significantly decreased in CD36i group compared with scrambled siRNA (NCi) group (P<0.01). The mRNA levels of CD11b and CD80 were decreased in CD36i group compared with NCi group (P<0.01). The cell surface area and cellular adhesiveness were increased in CD36OE group compared with empty vector (vector) group (P<0.05). The mRNA levels of CD11b and CD80 were increased in CD36OE group compared with vector group (P<0.01). The phosphorylation levels of ERK and Src were decreased in CD36i group compared with NCi group (P<0.05). CONCLUSION CD36 promotes the differentiation of human monocyte THP-1 cells to macrophages by increasing the phosphorylation of Src and further activating ERK.     

Construction of lentiviral vector-mediated siRNA knockdown of ER-α36and its action on gastric cancer cell growth

AIM: To construct a lentiviral vector for stable delivery of the ER-α36gene and to detect its effect on SGC7901 cell growth. METHODS: The efficient RNAi targeting sequences identified for the ER-α36gene were screened. The Oligo DNA was synthesized with target sequences and annealed to form double-stranded DNA. Then it was digested by XhoI and EcoR I and connected with GV307 vector to produce LV-ER-α36-RNAi lentiviral vector. PCR was used to screen the positive clones and sequence. The LV-ER-α36-RNAi, pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into 293T cells for producing lentiviral vector and infecting SGC7901 cell line. Fluorescence microscopy, real-time PCR and Western blotting were used to detect the transfection efficiency and gene silencing effect. 17β-estrodial at concentration of 1×10-10 mol/L was used to stimulate the recombinant cell line, and the action on the growth of gastric cancer cells and the expression of Src, ERK1/2 and cyclin D1 were determined. RESULTS: DNA sequencing analysis confirmed the identity of recombinant shRNA expression vectors. Immunofluorescence assay demonstrated that transfection efficiency was above 80%. Transfection of LV-ER-α36-RNAi significantly knocked down the expression of ER-α36 at mRNA and protein levels with tetracycline (TeT) simulating as revealed by real-time PCR and Western blotting. Compared with control group, the growth of the recombinant cell line declined and the expression of Src, ERK1/2 and cyclin D1 and the activation of Src decreased (P<0.05).CONCLUSION: Lentiviral vectors that silenceER-α36expression are constructed successfully and can be used to study the role of ER-α36 in gastric cancer. The ER-α36is related with many kinds of cancer cell growth, including gastric cancer cells.     

Palmitate triggers inflammatory response by upregulating fatty acid translocase in THP-1 macrophages

AIM: To investigate the role of fatty acid translocase (FAT/CD36) on palmitate-induced inflammation in human monocyte-derived macrophage THP-1.METHODS: THP-1 cells were treated with palmitate (0, 0.1 and 0.2 mmol/L) for 24 h. Transwell chamber assay was used to examine the migration ability of THP-1 cells. The mRNA expression of CD36, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) was measured by real-time PCR. The protein levels of TNF-α and IL-6 in the supernatant of cultured cells were measured by ELISA. The protein level of CD36 was examined by Western blot. Small interfering RNA (siRNA) targeting CD36 (siCD36) was used to inhibit the expression of CD36 in the THP-1 cells, and the changes of the cell migration and inflammatory response were monitored as mentioned above. RESULTS: Palmitate increased the expression of CD36 in the THP-1 cells (P<0.05). Palmitate also up-regulated inflammatory cytokine and chemokine levels, and the differences were statistically significant (P<0.05). Compared with control group, palmitate promoted migration of THP-1 cells. siCD36 was transfected into the THP-1 cells and the silencing efficiency was approximately 54%. The protein levels of TNF-α and IL-6 were also decreased in siCD36 group compared with scrambled RNA (scrRNA) group, and the differences were statistically significant (P<0.05). The migrated cells in siCD36 group were significantly less than those in scrRNA group (P<0.05).CONCLUSION: Palmitate promotes migration ability and triggers inflammatory response in the THP-1 macrophages by upregulating CD36 expression.     

Effect of ATP on proliferation and membrane protein expressionini mmortalized human fibroblasts

AIM: To investigate the effect of ATP on proliferationand membrane protein expression in immortalized human fibroblasts. METHODS: Applying the normal humanfi broblast cell line TIG-7 and OUMS-36, the immortalized human fibroblast cell line KMST-6 and SUSM-1 to culture for 24 hours and 96 hours with the different concentrations of ATP, ADP and AMP, respectively. The number of cell alive, DNA synthesis and the expressionof [³²P]-ATP labeled proteins in the membrane fraction were also observed. RESULTS: After 96 hours culture, the proliferation inhibition in immortalized cells exposed to 0.4 mmol/L ATP was 77%, while that of normal OUMS-36 cells treated withthe same concentration of ATP was 41%; the DNA synthesis of the immortalized cells exposed to 0.4 mmol/L ATP was greater suppressed than that of normal OUMS-36 cells. ADP was the only reagent tested thatreduced the number of cells significantly(P<0.01). When the immortalized cells exposed to ADP, AMP, adenosi ne, and phosphoric acid. In the presence of 1 mmol/L ATP, many KMST-6 cells died, but normal OUMS-36 cellsdid not. Compared with normal cells, the expresson of [³²P]-ATP labeled 30 kD, 31 kD, 33 kD and 40 kD proteins were markedly higher in the immortalized cells. CONCLUSION: ATP inhibited the proliferation and increased membrane protein expression in immortalized human fibroblasts.     

Effects of hypoxia on the expression of heparanase and invasiveness of SKOV3 ovarian cancer cell line

AIM: To evaluate the effects of hypoxia on the expression of heparanase and the invasiveness of SKOV3 ovarial carcinoma cell line. METHODS: SKOV3 cells were incubated at either normoxia (37 ℃, 5％CO2, 21％O2) or hypoxia (37 ℃, 5％CO2, 1％O2) condition for 12 h, 24 h and 36 h. RT-PCR and Western blotting were used to detect mRNA and the protein expressions of heparanase under different conditions. Cell invasiveness was measured by matrigel invasion assay. RESULTS: Compared to normoxia group, the heparanase mRNA expression level in hypoxia group was increased and in 12 h hypoxia group was the highest. The heparanase protein expression in hypoxia group was also significantly increased (P<0.01) and the expression of heparanase in hypoxia group was also different (P<0.05). Compared to normoxia group, the level of cell invasion was markedly increased in 12 h, 24 h and 36 h groups (P<0.05). During 12-36 h hypoxia period, the increase in hypoxia-induced invasiveness in SKOV3 cell line showed a time-dependent manner (P<0.05). Meanwhile, there was a positive correlation between the expression of HPA and the invasiveness of SKOV3 cells (r=0.8530, P<0.05). CONCLUSION: Invasion of SKOV3 cells in hypoxia condition correlates with heparanase level. Hypoxia plays an important role in the augmentation of the heparanase expression and the invasiveness of human ovarian cancer.     

APS improves free fatty acid metabolism by activating AMPK and promoting translocation of FAT/CD36 in C2C12 myoblasts

AIM：To investigate the effect of Astragalus polysaccharides (APS) on the metabolism of free fatty acids (FFAs) in C2C12 myoblasts. METHODS：Cultured C2C12 myoblasts were used in the study. The viability of C2C12 myoblasts treated with FFAs at different concentrations for different time was observed by MTT assay. The concentrations of FFAs in the medium were detected by acetyl-CoA synthase (ACS)-acetyl-CoA oxidase (ACOD) method. The expression of fatty acid translocase (FAT/CD36), AMPK and p-AMPK protein was examined by Western blotting. RESULTS：FFAs decreased the viability of C2C12 myoblasts in a time- and concentration-dependent manner. Compared with FFAs group, the expression of cellular membrane FAT/CD36 and p-AMPK proteins increased in FFAs+APS group, but total AMPK and FAT/CD36 protein expression was not significantly changed. Meanwhile, the concentration of FFAs in the medium decreased and the cell viability increased in FFAs+APS group as compared with the group. CONCLUSION：APS improves the metabolism of FFAs by activating AMPK and promoting translocation of FAT/CD36 in C2C12 myoblasts.     

Mouse melanoma cell exosomes promote expression of Rac1 protein in fibroblasts

AIM To investigate the effect of exosomes secreted by mouse melanoma cells on the expression of Ras-related C3 botulinum toxin substrate 1 (Rac1) protein in fibroblasts. METHODS Ultracentrifugation was adopted to separete exosomes secreted by mouse melanoma B16-F10 cells. The morphological structure of exosomes was observed by negative-staining electron microscopy. The size distribution of exosomes was determined by nanoparticle tracking analysis (NTA). The exosomal markers, tumor susceptibility gene 101 (Tsg101) and tyrosinase-related protein 2 (Tyrp2), were identified by Western blot. Laser confocal microscopy was used to observe the process that mouse embryonic fibroblasts (MEF) took in exosomes during co-culture. Immunocytochemical staining and Western blot were used to detect the expression of Rac1 protein in MEF. RESULTS B16-F10 cell exosomes showed a typical tea tray-like structure, with a size range of 141~255 nm, and expressed protein markers Tsg101 and Tyrp2. The results of laser confocal microscopy showed that compared with co-culture at 0 h, a small number of exosomes appeared in the MEF at 12 h, and a large number of exosomes accumulated in the MEF after co-cultured for 24 and 36 h. Western blot analysis showed that compared with co-culture at 0 h, the expression of Rac1 protein in the MEF was significantly increased at 24 h and 36 h of co-culture (P<0.01). The results of immunocytochemical staining showed that compared with co-culture at 0 h, the positive expression level of Rac1 in the MEF cells was significantly increased at 12 h, 24 h and 36 h of co-culture (P<0.05 or P<0.01). CONCLUSION Intake of exosomes secreted by mouse melanoma cells promotes the expression of Rac1 protein in fibroblasts.     

Effects of chronic EECP on protein expression in thoracic aorta endothelial cells in porcine with hypercholesteremia

AIM: To explore the effect of chronic enhanced external counterpulsation (EECP) on the protein expression in thoracic aorta endothelial cells in porcine with hypercholesteremia by proteomic techniques.METHODS: After performed EECP for 36 h on the hypercholesteremia porcines, the endothelial cells (ECs) were collected from the thoracic aorta and analyzed with proteomic techniques.RESULTS: Six kinds of protein were detected in ECs in the EECP group, but not in the hypercholesteremia control group.CONCLUSION: Chronic EECP may protect ECs against damage resulted from hypercholesteremia by regulating protein expression in artery ECs, subsequently improving cell adhesion and intracellular lipid metabolism and reducing ECs apoptosis.     

miR-126 regulates expression of vascular endothelial growth factor in EA.hy926 cells

AIM：To explore the effects of miR-126 on the expression of vascular endothelial growth factor (VEGF) in vascular endothelial cell line EA.hy926. METHODS：EA.hy926 cells were cultured in vitro and transfected with miR-126 mimics or miR-126 inhibitor by cation-mediated transfection method. The total RNA was extracted from the culture cells 36 h after transfection of miR-126 mimics or miR-126 inhibitor. The expression of VEGF at mRNA and protein levels was detected by real-time PCR and Western blotting. RESULTS：Thirty-six hours after transfection of miR-126 inhibitor at concentration of 50 nmol/L, the expression of VEGF at mRNA and protein levels increased significantly as compared with the negative control (P<001). However, transfection of miR-126 mimics at concentration of 50 nmol/L for 36 h significantly decreased the expression of VEGF at mRNA and protein levels as compared with the negative control. CONCLUSION：miR-126 inhibits the expression of VEGF. VEGF may be one of the target genes regulated by miR-126.     

Effects of andrographolide on invasion and apoptosis of ovarian cancer SKOV-3 cells

AIM:To investigate the effects of andrographolide on the invasion and apoptosis of ovarian cancer cell line SKOV-3,and to explore the possible mechanisms.METHODS:SKOV-3 cells were treated with different concentrations (0,5,10,20 or 40 μmol/L) of andrographolide for different time (12,24,36 or 48 h),and then the cell viability was determined by CCK-8 assay.The cell invasion ability was analyzed by Transwell assay and cell apoptosis was detected by TUNEL staining.The protein levels of p-PI3K,p-Akt and p-mTOR were examined by Western blot.RESULTS:The results of CCK-8 assay revealed that andrographolide inhibited the growth of SKOV-3 cells in a dose-and time-dependent manner.Treatment with andrographolide at 20 μmol/L for 36 h significantly decreased the invasion ability of SKOV-3 cells,while increased cell apoptosis.In addition,the protein levels of p-PI3K,p-Akt and p-mTOR were reduced after andrographolide treatment.CONCLUSION:Andrographolide inhibits the growth and invasion of ovarian cancer SKOV-3 cells by suppression of PI3K/Akt/mTOR signaling pathway.     

Relationship between histone H3K36 trimethylation and acute renal injury induced by ischemia/reperfusion

AIM: To observe the expression change of H3K36 trimethylation (H3K36me3) in acute kidney injury (AKI) induced by ischemia/reperfusion (IR) and analyze its correlation with SMYD2 and renal injury, and to provide a new target for clinical treatment of IR-AKI. METHODS: The ICR mice (n=30) were randomly divided into IR group (n=15) and sham operation group (sham group, n=15). The AKI model was established by clamping bilateral renal pedicles for 45 min. The serum and renal tissues of the mice were collected after the model was established for 24 h. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by biochemical method. The pathological changes of the kidney were observed by HE staining. The changes of NGAL and cleaved caspase-3 were observed by immunohistochemical (IHC) staining. The protein levels of NGAL, SMYD2, H3K36me3, p-P53, P53, cleaved caspase-3, Bax, Bcl-2, STAT3, p-STAT3, JNK and p-JNK1/2/3 in the renal tissues were determined by Western blot. The relationship between H3K36me3 and SMYD2/NGAL was analyzed by Pearson correlation method. RESULTS: Compared with sham group, BUN and SCr levels of the mice in IR group were significantly increased (P<0.05). HE staining showed significant edema, abscission and necrosis in renal tubular epithelial cells of the micc in IR group, and abscission of brush border and large amount of cell debris in the lumen were also observed. The IHC staining results showed that the protein levels of NGAL and cleaved caspase-3 in IR group were significantly increased. The results of Western blot showed that the protein levels of NGAL, SMYD2, H3K36me3, p-P53, cleaved caspase-3, Bax, STAT3, p-STAT3, JNK and p-JNK1/2/3 in IR group were significantly up-regulated (P<0.05), while the Bcl-2 levels were significantly down-regulated (P<0.05). The correlation analysis showed that H3K36me3 was positively correlated with the levels of SMYD2 and NGAL. CONCLUSION: H3K36me3 is closely related to SMYD2 and renal injury in IR-AKI mice, and the up-regulated expression of H3K36me3 may be involved in the regulation of IR-AKI occurrence and development together with the activation of STAT3 and JNK signaling pathways.     

Effect of KLF6 gene on nucleotide excision repair capacity in human hepatocellular carcinoma cell line HepG2

AIM: To investigate the roles of KLF6 gene on nucleotide excision repair capacity of human hepatocellular carcinoma cell line HepG2. METHODS: Expression of KLF6 was detected by RT-PCR and Western blotting, followed by HepG2 cells treated with cisplatin (5 mg/L). Host cell reactivation assay was used to assess nucleotide excision capacity of cisplatin-damaged HepG2 cells.RESULTS: Upregulation of KLF6 mRNA or protein in HepG2 cells treated with 5 mg/L concentration of cisplatin was in a time-dependent manner, but KLF6 protein was degraded when exposed to cisplatin at same dose (5 mg/L) for 36 h. HCR assay showed that NER capacity of KLF6 overexpressing HepG2 cells was significantly enhanced, compared to that in control cells. CONCLUSION: DNA damage induced by cisplatin induces the expression of KLF6 gene. KLF6 gene may play an important role in mechanism of nucleotide excision repair in HepG2 cells.     

Oxidized low-density lipoprotein induces autophagy in macrophages via CD36-mediated oxidative stress

AIM: To investigate the effect of oxidized low-density lipoprotein (ox-LDL) on autophagy in macrophages and the underlying molecular mechanisms. METHODS: RAW264.7 macrophages were pretreated with 2 mg/L anti-CD36 monoclonal antibody (anti-CD36 mAb), 5 μmol/L diphenyleneiodonium (DPI), 3 mmol/L 3-methyladenine (3-MA) or 1 μmol/L rapamycin for 1 h and then treated with ox-LDL (100 mg/L) for 12 h. The viability of the cells was measured by MTT assay. The activities of lactic dehydrogenase (LDH) in the medium and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase (SOD) in the cells as well as the levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) were determined to characterize the membrane integrity and the oxidative stress, respectively. The protein levels of beclin-1 and microtubule-associated protein 1 light chain 3-II (LC3-II), 2 important molecular markers of autophagy, were examined by Western blotting. RESULTS: ox-LDL induced autophagy in RAW264.7 macrophages as assessed by upregulation of beclin-1 and LC3-II. Similar to 3-MA, an autophagy inhibitor, anti-CD36 mAb significantly inhibited the ox-LDL-induced upregulation of beclin-1 and LC3-II. Anti-CD36 mAb suppressed the ox-LDL-induced oxidative stress as revealed by decreased NADPH oxidase activation, ROS and MDA generation as well as increased SOD activity. Similar results were observed in the cells pretreated with DPI, a NADPH oxidase inhibitor. Moreover, DPI significantly inhibited the ox-LDL-induced upregulation of beclin-1 and LC3-II. Inaddition, the decrease in the cell viability and increase in LDH release induced by ox-LDL were promoted by 3-MA and blocked by rapamycin (an autophagy inducer). CONCLUSION: ox-LDL induces autophagy in RAW264.7 macrophages, which may be involved in CD36-mediated ox-LDL uptake and subsequent activation of oxidative stress, and moderate activation of autophagy may protect macrophages from ox-LDL-induced injury.     

Shenmai injection improves insulin resistance in 3T3-L1 cells

AIM: To discuss the effect of Shenmai injection on insulin resistance (IR) in 3T3-L1 cells and its mechanisms. METHODS: 3T3-L1 preadipocytes were induced by chemical reagents to differentiate into fully differentiated adipocytes. Oil red O staining was used to detect the differentiation level of the adipocytes. The insulin-resistant 3T3-L1 cell model was demonstrated using insulin, which was confirmed by glucose concentration in cell supernatant. The IR cell model was given 10 μmol/L rosiglitazone, 25 and 50 g/L Shenmai injection and normal saline for comparison. MTT assay was used to assess the cell activity of 3T3-L1 cells which was treated with drugs for 8, 16, 24 and 36 h. Glucose oxidase method was used to detect the glucose concentration in the cell supernatant at 8, 16 and 24 h. The protein levels of glucose transporter-4 (GLUT4), phosphatidylinositol 3-kinase (PI3K), AKT and p-AKT were determined by Western blot. RESULTS: 3T3-L1 adipocytes were successfully induced as shown by the positive oil red O staining. The IR cell model was demonstrated, and glucose concentration in the cell supernatant after treatment with Shenmai injection showed that Shenmai injection reduced the IR in 3T3-L1 cell model. The protein levels of GLUT4, PI3K and p-AKT increased compared to control group. CONCLUSION: Shenmai injection reduces the IR in 3T3-L1 cell model, which functions by increasing the protein levels of GLUT4, PI3K and p-AKT.     

Expression and purification of re-constructed human CRP and observation of its internalization into HeLa cells

AIM: To construct prokaryotic expression vector for human C-reactive protein (CRP), to acquire the functional fusion protein purified from BL21(DE3) transformed with vector pET14b/EGFP-hCRP, and to observe the internalization of the fusion protein his-EGFP-CRP into tumor cell line HeLa. METHODS: CRP gene sequence was amplified with the vector p91023/CRP as template by PCR, and was inserted into vector pET14b/MCS-EGFP-(N)36 to construct prokaryotic expression vector. The E. coli cells BL21(DE3) transformed with the re-constructed vector pET14b/EGFP-hCRP was induced by isopropyl-β-D-thiogalactopyranoside (IPTG), and the expressed protein his-EGFP-CRP were purified with affinity chromatography method and refolded with gradient filtration. The HeLa cells were observed under the fluorescence microscopy after the addition of purified renature protein. RESULTS: The results of identification by PCR, digestion with restriction endonuclease and sequencing indicated the construction of vector pET14b/EGFP-hCRP was correct; the SDS-PAGE showed that the transformed E. coli cells could be induced to express the fusion protein his-EGFP-CRP and the purification of proteins were successful. We could found fluorescent signal around the cell membranes, in the cytoplasm and nuclei in the observation of the HeLa cells incubated with his-EGFP-CRP. CONCLUSION: The prokaryotic expression vector for human CRP linked with his and EGFP coding sequence is successfully constructed. The fusion protein his-EGFP-CRP is purified and refolded. The reconstructed protein expressed by prokaryotic cells adheres to the membrane of tumor cell HeLa and is internalized into the cytoplasm and nuclei of the cells.     

Effect of quercetin on ox-LDL-induced lipid accumulation and peroxidation in mouse macrophages

AIM：To investigate the effect of quercetin (QUE) preconditioning on oxidized low-density lipoprotein (ox-LDL)-induced lipid accumulation and peroxidation in mouse RAW264.7 macrophages and the underlying molecular mechanisms. METHODS：RAW264.7 cells were pretreated with different concentrations (20, 40 and 80 μmol/L) of QUE for 30 min and then treated with ox-LDL (100 mg/L) for 24 h. Intracellular lipid droplets were assayed by oil red O staining. Extracellular lactate dehydrogenase (LDH) and malondialdehyde (MDA) and intracellular reactive oxygen species (ROS) were determined to characterize the membrane integrity and the lipid peroxidation, respectively. The mRNA and protein levels of CD36, an important scavenger receptor which mediates ox-LDL uptake, were examined by real-time PCR and Western blotting, respectively. RESULTS：Pretreatment with QUE (20, 40 and 80 mmol/L) significantly attenuated ox-LDL-induced lipid accumulation in RAW264.7 cells and foam cell formation in a dose-dependent manner. Ox-LDL induced LDH release in RAW264.7 cells. This cytotoxic effect was significantly inhibited by QUE pretreatment. Compared with ox-LDL group, the intracellular ROS content and MDA level in culture medium decreased markedly in QUE group. In addition, pretreatment with QUE attenuated ox-LDL-induced up-regulation of CD36 at mRNA and protein levels. CONCLUSION: QUE inhibits ox-LDL-induced lipid accumulation and peroxidation in mouse macrophages and the mechanism may partially involve its ability to down-regulate CD36 expression.     

Role of FAT/CD36 in high-fat diet-induced adipose tissue inflammation

AIM: To investigate the role of fatty acid translocase/CD36 (FAT/CD36) in adipose tissue inflammation induced by a high-fat diet. METHODS: C57BL/6J mice were fed with a normal-chow diet (NCD) or a high-fat diet (HFD) for 14 weeks. The content of free fatty acid (FFA) in the serum was measured by ELISA. The expression of CD36, cytokines and chemokines at mRNA and protein levels in the adipose tissues was determined by real-time polymerase chain reaction and Western blotting. Immunohistochemical staining was used to examine the macrophages infiltration in the adipose tissues. The inflammatory responses in CD36 knockout mice and wild type mice with high-fat diet were analyzed. RESULTS: The levels of FAT/CD36 were higher in HFD group than that in NCD group. HFD feeding enhanced the mRNA and protein expression of IL-1β, IL-6, TNF-α, MCP-1 and MIP-1, as well as promoted macrophage infiltration in the adipose tissues. Interestingly, as fed with HFD, the expression of cytokines/chemokines and macrophage infiltration were significantly reduced in adipose tissues of the CD36 knockout mice, compared with the wild type mice. CONCLUSION: High-fat diet promotes adipose tissue inflammation in the mice in a FAT/CD36-dependent manner.     

Effects of extracellular potassium on expression of HERG gene nonsense mutant L539fs/47

AIM: To study the effects of extracellular potassium on the protein expression of wild- type HERG and its mutant L539fs/47. METHODS: Wild-type HERG (WT) or its mutant HERG-L539fs/47 (MT) were transfected into HEK293 cells for 36 h. The cells were incubated in different media containing 0.8, 4.3 or 10 mmol/L potassium. After 6 h of incubation, the protein expression of HERG was detected by flow cytometry.After 12 h of incubation, the localization and quantity of the proteins were detected by laser confocal imaging and Western blot. RESULTS: Different from the retention of mutant protein in cytoplasm, wild-type HERG protein was mainly distributed in the cell membrane. The 2 proteins both increased with the changes of extracellular potassium. Flow cytometry showed that the fluorescence in the 2 groups both increased with the changes of extracellular potassium (P < 0.01). The fluorescence in WT group was significantly higher than that in MT group (P < 0.01). Western blot showed that mutant HERG protein included only one 60 kD band, different from the 135 kD and 155 kD bands in wild-type HERG, which were affected by the changes of extracellular potassium (P < 0.05). CONCLUSION: The retention of HERG mutant L539fs/47 protein in the cytoplasm is more than wild-type HERG. Chronic high extracellular potassium keeps the stability of wild-type and mutant HERG proteins on the cell membrane. Chronic low potassium reduces the expression of HERG channel proteins in a time-dependent manner.