Relationship between CD36 and cell proliferation and migration in hepatocellular carcinoma and its effect on human hepatocellular carcinoma cell xenograft models in nude mice / 肿瘤研究与临床

Objective:To observe the expression of CD36 in hepatocellular carcinoma tissues and cell lines, and to investigate the effects of CD36 on the proliferation and migration abilities of human hepatocellular carcinoma cell lines and human hepatocellular carcinoma cell xenograft models in nude mice.Methods:Differences in the expression levels of CD36 transcripts in 371 hepatocellular carcinoma and paracancerous tissues were analyzed based on information from The Cancer Genome Atlas (TCGA) database. Cancer tissues and corresponding paracancerous tissues of 48 hepatocellular carcinoma patients who were diagnosed and underwent surgical treatment at the Affiliated Hospital of Yangzhou University from January 2019 to February 2021 were prospectively collected, and the levels of CD36 mRNA in the tissues were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) method. Western blotting was used to detect CD36 protein levels in human hepatocellular carcinoma cell lines Huh7 and HCCLM3 and human normal liver cell line LO2. Plasmids containing CD36 interfering sequences and empty plasmids were transfected into Huh7 cells or HCCLM3 cells for sh-CD36 group and control group, respectively. The CCK-8 assay was used to detect the proliferation ability (expressed as absorbance value) of cells in each group at 0, 12, 24, 36, 48 and 60 h of culture, and the scratch healing assay and Transwell assay were used to detect the migration ability of cells in each group. The Huh7 cells of sh-CD36 group or control group were injected into the axillary subcutis of BALB/c nude mice, with 4 mice in each group, to construct nude mice models of human hepatocellular carcinoma xenografts; the long and short diameters of tumor were measured weekly after 1 week of inoculation, and the tumor volume was calculated. The nude mice were put to death after 5 weeks of inoculation, and the tumor specimens were collected and weighed; the tumor cell morphology was observed under the microscope, and the expressions of CD36 and Ki-67 proteins in the tumor tissues was detected by immunohistochemistry (IHC).Results:Analysis of the data from the TCGA database showed that the level of CD36 transcripts was higher in hepatocellular carcinoma tissues compared with that in paracancerous tissues (4.2±1.8 vs. 3.2±1.5, t = 2.28, P = 0.035). Tissues detection using qRT-PCR in 48 patients with hepatocellular carcinoma showed that the relative expression of CD36 mRNA in hepatocellular carcinoma tissues was higher than that in paracancerous tissues (0.76±0.26 vs. 0.48±0.23, t = 3.52, P < 0.001). Western blotting assay showed that CD36 protein level in Huh7 and HCCLM3 cells was higher than that in LO2 cells, which were (1.42±0.11) times and (1.68±0.16) times higher than LO2 cells, respectively (both P < 0.001). At the mRNA and protein levels, the CD36 of Huh7 and HCCLM3 cells in the sh-CD36 group was lower than that in the corresponding control group (both P < 0.001). CCK-8 assay showed that the proliferative ability of Huh7 cells and HCCLM3 cells in the sh-CD36 group was lower than that in the corresponding control group after 36 and 24 h of culture (both P < 0.01). Scratch healing assay showed that the scratch healing rates of Huh7 cells [(12±3)% vs. (30±5)%, t = 4.01, P < 0.001] and HCCLM3 cells [(15±4)% vs. (29±5)%, t = 4.16, P < 0.001] in the sh-CD36 group were lower than those in the corresponding control group at 48 h of culture; Transwell assay showed that the number of Huh7 cells [(46±6) cells/field of view vs. (88± 6) cells/field of view, t = 5.56, P < 0.001] and HCCLM3 cells [(42±5) cells/field of view vs. (82±7) cells/field of view, t = 5.34, P < 0.001] penetrating into the membrane in 24 h in the sh-CD36 group was less than that in the corresponding control group. Five weeks after subcutaneous injection, the tumor volume [(682±268) mm 3vs. (1 375±512) mm 3, t = 4.73, P = 0.006] and tumor mass [(432±95) mg/mouse vs. (871±109) mg/mouse, t = 6.57, P < 0.001] of nude mice injected with Huh7 cells of the sh-CD36 group were lower than those of nude mice injected with Huh7 cells of the control group; under the microscope, the density of tumor cells in transplanted tumor specimens of nude mice injected with Huh7 cells of the sh-CD36 group was lower than that in nude mice injected with Huh7 cells of the control group, and the expression levels of both CD36 and Ki-67 proteins were also low. Conclusions:CD36 expression is up-regulated in cancer tissues of hepatocellular carcinoma patients and human hepatocellular carcinoma cell lines Huh7 and HCCLM3, and it may associate with cell proliferation and migration of hepatocellular carcinoma. Knockdown of CD36 expression significantly inhibits the proliferation and migration abilities of hepatocellular carcinoma cells in vitro, and inhibits the tumors of human hepatocellular carcinoma cell xenograft models in nude mice.

Molecular docking in Nεcarboxymethyl lysine targeting scavenger receptor CD36 / 中华老年心脑血管病杂志

Objective To study whether Nεcarboxymethyl lysine(CML)can form a good molecular docking with the scavenger receptor CD36and induce a stable interaction.Methods The interaction between CML and CD36was studied by co-immunoprecipitation.The binding mode and affinity of CD36to CML were tested using AutoDock 4.2,iBabel and XQuartz-2.7.7software respectively. Results Co-immunoprecipitation showed that anti-CD36antibody magnetic bead could precipitate CD36from the total protein in RAW264.7cells and anti-CML could detect CD36 binding CML.CD36had a good molecular docking with CML,CD36and CML interacted stably with each other.The affinity of CML to 4Q4Bprotein structure of CD4extracellular domain was -29.62kJ/mol.ARG82,ASN71and THR70were the products of amino acid receptor interaction. Further docking analysis showed that CML could form 3interacting hydrogen bonds with 4Q4B,and the docking prediction inhibition constant was 6.92with a root mean square deviation of 2.54.Conclusion A good molecular docking between CML and 4Q4Bprotein structure of CD36extracellular domain can induce a stable interaction between CML and CD36.Hydrogen bonding is the main interaction mode.

CD36 expression in monocytes and ankylosing spondylitis / 中国组织工程研究

BACKGROUND:Ankylosing spondylitis is an autoimmune disease involved in chronic systemic inflammation. Tumor necrosis factor and interleukin-6 levels increased in patients with ankylosing spondylitis. Inflammatory factors such as tumor necrosis factor and interleukin-6 can suppress CD36 expression in monocytes. OBJECTIVE: To analyze the correlation between CD36 expression in monocytes and ankylosing spondylitis. METHODS:A total of 84 newly diagnosed ankylosing spondylitis patients and 111 healthy individuals were included in this study. CD36 expressions in monocytes in ankylosing spondylitis patients and healthy individuals were tested using flow cytometer; meanwhile, biochemistry, immunology, routine blood examination and related inflammatory markers were determined between the two groups. RESULTS AND CONCLUSION:Results of baseline data in both groups demonstrated that CD36 fluorescence intensity in monocytes was significantly lower in patients with ankylosing spondylitis compared with healthy controls (P < 0.01). CD36 fluorescence intensity in monocytes was negatively correlated with C-reactive protein, erythrocyte sedimentation, interleukin-6 and tumor necrosis factor. In addition, CD36 fluorescence intensity in monocytes was negatively correlated with BASDAI score. Logistic regression analysis showed that erythrocyte sedimentation, interleukin-6, tumor necrosis factor and CD36 fluorescence intensity in monocytes were associated with ankylosing spondylitis, and risk factors for ankylosing spondylitis (P < 0.05). These findings confirm that inflammatory cytokine in patients with ankylosing spondylitis weakened the expression of CD36 in monocytes. There was a remarkable association between low expression of CD36 expression in monocytes and ankylosing spondylitis. CD36 expression of monocytes clinicaly may be considered to be an effective indicator to evaluate inflammation and disease activity in patients with ankylosing spondylitis.

Development of a duplex fluorescence RT-PCR assay for identifying SRBⅠgene knockout mice / 天津医药

Objective To develop a duplex fluorescence RT-PCR assay for detection of scavenger receptor class B, typeⅠ(SRBⅠ) knockout mice. Methods Primers and probes were designed according to knockout region of SRBⅠgene and related substituted sequence. DNA samples were extracted from tails of mice and performed amplification using real-time PCR. SRBⅠgenotypes of mice were analyzed according to amplification curves of FAM and CY5 channels. Finally, the sensitivity of the method was detected and the accuracy was verified by the direct sequencing. Results The homozygous SRBⅠwild genotype showed an amplification curve only in FAM channel. When the homozygous SRBⅠknockout genotype was present, the typical S amplification curve appeared only in the CY5 channel. Heterozygous genotype showed two typical S amplification curves in both FAM and CY5 channels, respectively. The results showed that the sensitivity reached 4×101 copies/μL, and there was complete concordance between this method and direct DNA sequencing. Conclusion The new method is simple, rapid and accurate, which is suitable for genotyping SRBⅠknockout mice.

The Association of CD36 rs7755 and rs3211956 Single Nucleotide Polymorphism with Premature Coronary Heart Disease / 天津医药

Objective To investigate the risk factors of premature atherosclerotic three-vessel coronary artery dis?ease (CHD), and the association between single nucleotide polymorphism (SNP) of CD36 rs3211956, rs7755 and premature CHD. Methods Patient with premature three-vessel coronary artery disease (n=102) which were confirmed by consecutive coronary angiogram (lesion group) and patients (n=72) without CHD (control group) were enrolled in the study. Conventional CHD risk factors were compared between the two groups as well as SNPs of CD36 rs3211956 and rs7755 to disclose inde?pendent risk factor for CHD, which were measured by mass spectrometry. Results Among the conventional CHD risk fac?tors, male, HBP, high LDL-C, low HDL-C were independent risk factors of premature CHD. The GT genotype proportion of rs3211956 was significantly lower (χ2=8.042,P=0.005) in the lesion group than that in control group while the TT genotype proportion is significantly higher in lesion group compared with that in control group (χ2=6.191,P=0.014). Patients with the TT genotype have higher score of BMI than patients with GG or GT genotype (P=0.037). The G allele proportion of rs7755 in the lesion group was significantly higher than control group (χ2=3.636, P=0.047). Patients of the GG genotype have higher scores of BMI but lower level of HDL-C than those with AA or AG genotype (P<0.001). Logistic regression analysis re?vealed that after excluding a number of confounding factors, GG and TT genotype of rs3211956 and GG and GA genotype of rs7755 were respectively one of the independent risk factors for premature CHD. Conclusion The SNPs of CD36 rs7755 and rs3211956 may be the independent risk factors of premature coronary heart disease and might affect the the onset of CHD by affectting BMI and HDL-C.

The effects and underlying mechanism of CD36 in high glucose-induced rat glomerular mesangial cells apoptosis / 中华肾脏病杂志

Objective To investigate the effects and underlying mechanism of the scavenger receptor CD36 in high glucose-induced rat glomerular mesangial cells apoptosis.Methods The mesangial cells of rats were divided into 4 groups:control group (5.6 mmol/L glucose),mannitol group (24.2 mmol/L mannitol+5.6 mmo]/L glucose),high glucose group (30 mmol/L glucose),CD36 monoantibody group (30 mmol/L glucose+CD36 mono-antibody).The intracellular ROS level was detected by confocal microscopy with fluorescent probe CM-H2DCFDA.MDA,GSH-PX,8-OHDGA in cell supernatant were detected.Apoptosis was determined by flow cytometry followed by Annexin V-FITC/PI double stains.The expression of CD36,Bax and Bcl-2 were detected by RT-PCR and Western blotting.Results The expression of CD36 was detected in glomerular mesangial cells.The highest level was found in high glucose group in 24 hours.There was no significant difference found between control group and mannitol group with respect to intracellular ROS generation,MDA,8-OHDG,GSH-PX level,apoptosis rate,expression of CD36,Bax and Bcl-2 (all P ＞ 0.05).There was no significant difference in the expression of CD36 between CD36 mono-antibody group and high glucose group (P ＞ 0.05).Compared to control group,the intracellular ROS generation,MDA and 8-OHDG levels,apoptosis rate,the expression of CD36 and Bax were significantly increased,the GSH-PX level and the expression of Bcl-2 were significantly lower in high glucose group (all P ＜ 0.05).Compared to the high glucose group,the intracellular ROS generation,MDA and 8-OHDG levels,apoptosis rate,the expression of Bax were suppressed but the GSH-PX level and the expression of Bcl-2 increased in CD36 mono-antibody group (all P ＜ 0.05).The intracellular ROS level was positively correlated with apoptosis rate,protein expression of CD36 and Bax gene,was negatively correlated with Bcl-2 protein expression.Conclusions CD36 was involved in the high glucose induced apoptosis of mesangial cells which was potentially mediated by an increased level of oxidative stress.

Effects of High Density Lipoprotein on Expression of Alpha-Granulemembrane Protein (CD62P) and Lysosome Intact Membrane Protein (CD63) on Platelet / 天津医药

Objective To investigate the effects of high density lipoprotein (HDL) on thrombin-activated platelet al-pha-granulemembrane protein (CD62P) and lysosome intact membrane protein (CD63) expressions in vitro. Methods The equivalent volume of washed platelets prepared by hand was preincubated with HDL (1 g/L) in 37℃water for 15 minutes, which was then stimulated with different concentrations of thrombin (0.5 U/mL, 1 U/mL and 10 U/mL) for 10 minutes in wa-ter of 37℃. Meanwhile another three groups of washed platelets were incubated with thrombin (0.5 U/mL, 1 U/mL and 10 U/mL) for 10 minutes, respectively. The CD62P and CD63 from each sample were analyzed by flow cytometry (FCM). Results The CD62P positive rates of HDL-preincubated groups were significantly lower than those of different concentrations of thrombin groups (0.5 U/mL,1 U/mL and 10 U/mL) in the absence of HDL (11.55%± 1.34% vs 18.14%± 1.50%, 17.19%± 0.17% vs 26.24%± 0.77% and 19.79%± 0.32% vs 80.38%± 5.66%,P ＜ 0.01). Meanwhile, The CD63 positive rates of HDL-preincubated groups were also significantly lower than those of thrombin-treated (0.5 U/mL, 1 U/mL and 10 U/mL) groups without HDL, namely,2.92%±0.22%vs 8.09%±0.48%(P＜0.001), 4.20%±0.98%vs 14.15%±1.39%(P＜0.001) and 5.12%± 0.09% vs 24.48%± 1.71%(P ＜ 0.01). Conclusion HDL inhibits the expression of CD62P and CD63 on throm-bin-stimulated platelets in vitro.

CD36 signaling inhibits the translation of heat shock protein 70 induced by oxidized low density lipoprotein through activation of peroxisome proliferators-activated receptor gamma

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor gamma (PPAR gamma) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPAR gamma activity or knockdown of PPAR gamma expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPAR gamma through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPAR gamma siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress- induced gene expression by suppressing translation via activation of PPAR gamma in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPAR gamma.

Influences of wild-type p53 gene overexpression on the differentiation, apoptosis and expression of scavenger receptor CD36 in U937 cells / 中国病理生理杂志

AIM: To study the effect of wild-type p53 gene on the differentiation, apoptosis and expression of scavenger receptor CD36 in U937 cells. METHODS: Recombinant adenovirus vector with wild-type p53 gene was constructed and used to transfect U937 cells. With the expression of wild-type p53 gene following adenoviral infection, transfected U937 cells were largely promoted to differentiate into macrophages. RESUITS: Trypanblue-staining test demonstrated that the percentage of positive cells increased from (14.2±5.5)% to (64.6±9.2)% and nitroblue tetrazolium (NBT) reduction test reached similar results (6.3±1.8)% vs (49.7±12.6)%. Furthermore, CD36 mRNA was up-regulated as confirmed by RT-PCR. The increased expression level of CD 36 was also detected by flow cytometry analysis. CONCLUSION: These results suggest that wild-type p53 gene can affect U937 cells differentiation and apoptosis, up-regulate expression of scavenger receptor CD36. It may have a potential significance on atherogenesis.

Inflammatory stress induces insulin resistance in CD36 knockout mice / 第三军医大学学报

Objective To explore whether the inflammatory stress can increase insulin resistance in the CD36 knockout (KO) mice.Methods After the mice were fed a standard chow for 14 weeks,oral glucose tolerance test,insulin release tests,lipids metabolism,SAA,IL-6,TNF-? and hepatic mRNA and proteins expression of mTOR,S6K,IRS-1,pIRS-1,2 were measured.Results Compared with the wide-type,the CD36 KO mice un-inflamed exhibited insulin resistance,and the insulin resistance index was increased[(3.01?1.24) vs (0.81?0.12),P

Effect of activated nuclear receptors PPAR?,LXR?,RXR? on ABC1 and CD36 mRNA expression in THP-1 cell / 中国病理生理杂志

AIM: To study the discrepancy and it's significance of ATP binding cassette transporter(ABC1) and CD36 mRNA expression while activate nuclear receptor RRAR?,LXR?,RXR? in THP-1 cells. METHODS: Cultured THP-1 cells were induced to become macrophage by PMA at concentration of 40 ?g/L for 48 hours. Troglitazone, 22(R)-hydrocholesterol and 9-cis-retinoid acid(9-CRA),activators for PPAR?,LXR? and RXR? respectively, were incubated with the macrophage for 24 hours. Total RNA of these cells were abstracted and reverse transcriptional polymerase chain reaction were performed to inspect the expression of ABC1 and CD36 mRNA in the cells. RESULTS: Expression of ABC1 mRNA increased in the presence of troglitazone, 22(R)-HC and 9-CRA. CD36 mRNA also increased while PPAR? activated. Activation of LXR? showed almost no effect on CD36 mRNA expression, and expression of CD36 mRNA was decreased by 9-CRA.CONCLUSION: The discrepancies existing in ABC1 and CD36 mRNA expression while activate nuclear receptors PPAR?,LXR?,RXR? in THP-1 cells implyed that LXR?,RXR? were better targets to deal with to improve cholesterol reverse transportation.

Effects of extract of gingko biloba on the lipid metabolism and the function of macrophages from diabetic rats / 中国病理生理杂志

AIM: To study effect of extract of ginkgo biloba(EGb) on the lipid metabolism and the function of macrophages from diabetic rats.METHODS: Sprague-Dauley rats were divided into four groups: normal control group,high-fat group,diabetic group and EGb treatment group.At the end of experiment,the rats were sacrificed,the blood glucose,blood insulin and serum lipid were measured.The activity of superoxide dismutase(SOD),content of malondialdehyde(MDA),nitric oxide(NO) in alveolar macrophages(AM) and peritoneal macrophages(PM) were assayed.In addition,peroxisome proliferator activated receptor ?(PPAR?),CD36 mRNA expression in AM was measured by RT-PCR.RESULTS: The concentration of the blood glucose,blood insulin and total cholesterol(TC),total triglycerides(TG),low-density lipoprotein-cholesterol(LDL-C) in blood increased significantly in type 2 diabetic group.The supplement of EGb decreased blood glucose,blood insulin and TC,TG,LDL-C levels.The activity of SOD decreased,while the content of NO,MDA increased in the diabetic macrophages,the activity of SOD became increased,but the content of NO and MDA decreased in EGb-treated group.The mRNA expression level of CD36 and PPAR? in alveolar macrophages from diabetic group increased,while expression level of CD36 and PPAR? mRNA in EGb treated rats continued to rise.CONCLUSIONS: EGb corrected insulin resistance and ameliorated disturbance of lipid metabolism caused by type2 diabetes in rats.Adjustment of PPAR? and CD36 mRNA expression of as well as reduction of lipid peroxidation and NO level may be involved in this process.

Correlation between local hormones and CD_(36) transcription level in women with polycystic ovary / 中华妇产科杂志

Objective To detect CD_(36) expressions in polycystic ovary (PCO), and to explore its correlation with local androgen and insulin at transcription level. Methods From August 2002 to February 2003, 12 patients with asymmetric PCO, 15 primary or secondary infertile patients without endocrine disorders and 8 polycystic ovary syndrome (PCOS) with bilateral PCO were recruited. Extraction of follicular fluid and detection of testosterone(T), dehydroepiandrosterone sulfate (DHEAS), insulin (INS) and androstenedione (A_2) were performed. Relative CD_(36) mRNA expression level of human ovarian inner thecal cells was analyzed by auto image analysis system (IAS) after RT-PCR. Results The level of CD_(36) mRNA expression in thecal cells was 0.24?0.07 in polycystic ovary of PCO group and 0.21?0.05 in bilateral ovaries of PCOS group, respectively, which were significantly lower than 0.83?0.13 in normal ovaries (P