OBJECTIVE: To investigate the effect of thrombospondin-1 (TSP-1) on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism. METHODS: The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations. The early apoptosis and activity of caspase-3 were detected by flow cytometry. The effect of TSP-1 on the growth and differentiation of megakaryocytes was investigated by cell counting and CFU-MK culture. RESULTS: The flow cytometry and immunocytochemistry showed that CD36 antigen was expressed on the surface of Meg-01 cells. TSP-1 (5 μg/ml) inhibited the growth of Meg-01 cells, but had unobvious effect on M-07e cells. After addition of CD36 antibody FA6-152 (5, 10, and 25 μg/ml), the inhibition effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5, and 7.5 μg/ml) increased the positive expression of Annexin V (P<0.01) and caspase-3 activity (P<0.01), which indicated that TSP-1 had a significant effect on inducing apoptosis. After addition of CD36 antibody FA6-152 (25 μg/ml), the apoptosis induced by TSP-1 in Meg-01 cells was significantly reduced. TSP-1 (5, 10, and 25 μg/ml) could significantly inhibit the formation of CFU-MK in mouse bone marrow cells, while β-TG could not. CD36 antibody FA6-152 (25 μg/ml) could significantly reduce the inhibition of TSP-1 on CFU-MK. CONCLUSION TSP-1 may induce apoptosis of megakaryocytic leukemia cell line Meg-01 cells via CD36/caspase-3, which provides a potential new drug development and treatment target for clinical treatment of megakaryocytic leukemia.
Animals ; Apoptosis ; CD36 Antigens/metabolism* ; Caspase 3/metabolism* ; Cell Line ; Humans ; Leukemia, Megakaryoblastic, Acute ; Mice ; Thrombospondin 1/pharmacology*

OBJECTIVE: To analyze the molecular polymorphisms of CD36 among 58 blood donors with CD36 deficiency and compare with CD36 positive controls. METHODS: A total of 58 donors with CD36 deficiency during a screening conducted in the laboratory from September 2019 to December 2020 were enrolled as the test group, including 39 males and 19 females, while 120 platelet donors with CD36 positive were randomly selected as the controls, including 76 males and 44 females. All of the subjects were Han nationality. The PCR-SBT method was used to detect coding region of CD36 gene, and molecular mutations were compared with those CD36 positive controls. RESULTS: Among the 58 donors with CD36 deficiency, mutations appears in 32 individuals. The detection rate for type I was 71.43% (5/7), and type II was 51.92% (27/52), while among the 120 controls, mutations appears in 12 donors (10%). In the CD36 antigen-deficient donors, 16 variations were found, in which 329-330 del AC with the highest frequency accounted for 20.69%, followed by 1228-1239 del ATTGTGCCTATT(15.52%) and 1156 C>T(10.34%). Two variations, 198-205 del GATCTTTG and 220 C>T, led to premature termination of translation; four mutations, 329-330 del AC, 560 ins T, 1011-1049 39bp dupl and 1343-1344 ins TCTT, caused translation frame shift; 1228-1239 del ATTGTGCCTATT led to deletion of four amino acids (Ile-Val-Pro-Ile) at sites 410-413 of the peptide chain. The 1140 T>A and 1275 G>A were synonymous mutations, and the other 7 mutations resulted in the substitution of single nucleotide. The platelet expression in the donors of CD36 positive with 329-330 del AC or 1228-1239 del ATTGTGCCTATT mutation (heterozygote) was lower than those CD36 positive individuals without mutations (homozygote). CONCLUSION Multiple gene mutations in the CD36 coding region may cause CD36 deficiency, and the heterozygous individuals with mutations may lead to CD36 antigen reduction or deletion. Mutation is not detected in 44.83% of CD36 deficient individuals, there may be some other reasons for the CD36 antigen deficiency.
Blood Donors ; Blood Platelet Disorders/metabolism* ; Blood Platelets/metabolism* ; CD36 Antigens/metabolism* ; Female ; Genetic Diseases, Inborn ; Humans ; Male

The development of nonalcoholic fatty liver disease (NAFLD) is closely related to the fatty acid (FA) uptake. This study aimed to investigate the effect of Krüppel-like factor 9 (KLF9) on CD36 (typical fatty acid translocase), hepatocellular lipid metabolism as well as the development and progression of nonalcoholic fatty liver. High-fat diet-induced obese C57BL/6J mice and db/db mice were used to test the expression levels of Klf9 and Cd36 in the livers. The primary hepatocytes were isolated from C57BL/6J mice, treated with Ad-GFP, Ad-Klf9, Ad-shCtrl or Ad-shKlf9, and then incubated with oleic acid and palmitic acid for 24 h. Liver-specific knockout of Klf9 mice were established. The protein levels and relative mRNA levels were examined by Western blot and real-time PCR, respectively. Triglyceride content was determined by using an assay kit. Lipid content was determined by Oil Red O staining. The results showed that: (1) Klf9 expression levels were increased in the livers of high-fat diet-induced obese mice and db/db mice, compared to their respective control mice. (2) Adenovirus-mediated overexpression of Klf9 in primary hepatocytes increased Cd36 expression and cellular triglyceride contents. (3) In contrast, adenovirus-mediated knockdown of Klf9 expression in primary hepatocytes by Ad-shKlf9 decreased Cd36 expression and cellular triglyceride contents. (4) Finally, Klf9 deficiency decreased liver Cd36 expression and alleviated fatty liver phenotype of high-fat diet-induced obese mice. These results suggest that KLF9 can regulate hepatic lipid metabolism and development of NAFLD by promoting the expression of CD36.
Animals ; CD36 Antigens/metabolism* ; Diet, High-Fat ; Kruppel-Like Transcription Factors/metabolism* ; Lipid Metabolism ; Liver ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Non-alcoholic Fatty Liver Disease/metabolism* ; Oleic Acid/metabolism*

Cardio-cerebral vascular disease induced by atherosclerosis is a serious cause of human health. The pathogenesis of AS is very complex,and the oxidized low-density lipoprotein( ox LDL) induced foam cells formation is considered to be the most important cytological change in AS. Based on the definition of " TCM chemical biology",we clarified the chemical composition of Ilex hainanensis,the effective substances of I. hainanensis on the activity of anti-AS were screened. Then we found that saponin BF523 had the good inhibitory effect on foam cell formation. In this research,we studied the BF523 as the research object to clarify the molecular target of the active compound of I. hainanensis by foam cell formation model. The results showed that BF523 significantly inhibited the oxidation of ox LDL-induced macrophage foaming and decreased the lipid content in macrophages. BF523 had inhibited the phagocytosis of ox LDL in macrophages by reducing the mRNA and protein levels of scavenger receptor CD36,thereby inhibiting the occurrence and development of AS. These findings not only clarified the mechanism of the inhibition of foam cell formation by saponin BF523,but also provided a useful exploration for the enrichment of the theory of " TCM chemical biology".
Atherosclerosis ; CD36 Antigens ; metabolism ; Cells, Cultured ; Foam Cells ; cytology ; drug effects ; Humans ; Ilex ; chemistry ; Lipoproteins, LDL ; adverse effects

OBJECTIVE: To construct eukaryotic expression vectors for human platelet CD36 gene 220 C>T and 429+4insg variants and analyze their expressions in HEK293T cells. METHODS: RNA was isolated from human platelets and reversely transcribed into cDNA. Sequences of 220C>T and 429+4insg variants were derived by PCR amplification. The target sequence was ligated into a pcDNA3.1/V5-His-TOPO vector by TA cloning, which was transformed into TOP10 E. coli. Positive plasmids were screened by blue-white selection. After sequencing, plasmid DNA carrying 220C>T or 429+4insg variant was used to transfect HEK293T cells with the help of effectene. Expression of CD36 protein was then analyzed by flow cytometry and Western blotting. RESULTS: An eukaryotic expression vector pcDNA3.1/V5-His-CD36 (220C>T/429+4insg) was constructed by TA cloning. After transfected into HEK293T cells, the 220C>T and 429+4insg variants resulted in CD36 deficiency in HEK cells, which was confirmed by flow cytometry and Western blotting. CONCLUSION The 220C>T and 429+4insg variants can cause CD36 deficiency in human platelets. This system may be used for assessing the effect of 220C>T, 429+4insg, and other variants on the expression of CD36.
Blood Platelets ; CD36 Antigens ; Cloning, Molecular ; Escherichia coli ; Eukaryota ; Genetic Vectors ; HEK293 Cells ; Humans ; Plasmids ; Transfection

BACKGROUND/AIMS: Hepatic steatosis is caused by an imbalance between free fatty acids (FFAs) uptake, utilization, storage, and disposal. Understanding the molecular mechanisms involved in FFAs accumulation and its modulation could drive the development of potential therapies for Nonalcoholic fatty liver disease. The aim of the current study was to explore the effects of picroside II, a phytoactive found in Picrorhiza kurroa, on fatty acid accumulation vis-à-vis silibinin, a known hepatoprotective phytoactive from Silybum marianum. METHODS: HepG2 cells were loaded with FFAs (oleic acid:palmitic acid/2:1) for 20 hours to mimic hepatic steatosis. The FFAs concentration achieving maximum fat accumulation and minimal cytotoxicity (500 μM) was standardized. HepG2 cells were exposed to the standardized FFAs concentration with and without picroside II pretreatment. RESULTS: Picroside II pretreatment inhibited FFAs-induced lipid accumulation by attenuating the expression of fatty acid transport protein 5, sterol regulatory element binding protein 1 and stearoyl CoA desaturase. Preatreatment with picroside II was also found to decrease the expression of forkhead box protein O1 and phosphoenolpyruvate carboxykinase. CONCLUSIONS: These findings suggest that picroside II effectively attenuated fatty acid accumulation by decreasing FFAs uptake and lipogenesis. Picroside II also decreased the expression of gluconeogenic genes.
Fatty Acid Transport Proteins ; Fatty Acids, Nonesterified ; Hep G2 Cells* ; Lipogenesis ; Milk Thistle ; Non-alcoholic Fatty Liver Disease ; Phosphoenolpyruvate ; Picrorhiza ; Stearoyl-CoA Desaturase ; Sterol Regulatory Element Binding Protein 1

OBJECTIVETo analyse the cases of platelet transfusion refractoriness after received HLA-matched unrelated donor hematopoietic stem cell transplantation, to analyze and identify the phenotype and genotype of CD36 in both the patient and stem cell donor, as well as the characteristic of antibody induced platelet transfusion refractoriness, and to analyse the efficacy of matched CD36-deficiency platelets transfusions.

METHODSThe CD36 expression on platelet and monocyte was analyzed by flow cytometry (FCM) in both patient and donor. Polymerase chain reaction sequence-based typing (PCR-SBT) was used to analyze the exons sequence of CD36 and HPA. Fast monoclonal antibody-specific immobilization of platelet antigen (F-MAIPA) and FCM were used to identify platelet antibodies in the patient. Short tandem repeat polymerase chain reaction (STR-PCR) was applied to monitor engraftment evidence. The platelet level was monitored. CD36- deficiency donor's platelets were selected from CD36- deficiency donor blood bank.

RESULTSThe donor was CD36 positive and the patient was typed I CD36 deficiency. The anti-CD36 antibody was identified in patient's serum (after transplantation), while the HLA and HPA-related antibodies were excluded. Sequence analysis of CD36 exon in the patient showed Exon 6 -1G>C(Change in splicing site) homozygote, which was a novel CD36 mutation. STR, HPA and CD36 of the patient (complete chimerism) were conversed to that of donor gene types on day 18 after allo-HSCT. The positive CD36 expression on platelet and monocyte in the patient was observed on day 96 after allo-HSCT. The patient showed the platelet transfusion refractoriness which was significantly improved after platelets transfusions from CD36 deficiency donors.

CONCLUSIONStem cell transplants resulted in anti-CD36 and caused platelet transfusion refractoriness, that was first reported in China. To ensure the efficacy of platelet transfusion, the CD36-deficiency patient should receive CD36 deficiency platelets for transfusion.

Antigens, Human Platelet ; Blood Platelet Disorders ; Blood Platelets ; CD36 Antigens ; China ; Humans ; Platelet Transfusion ; Thrombocytopenia

CD36 is a transmembrane glycoprotein, a multi-ligand receptor, possesses various biological functions. CD36 deficiency may stimulate the body to produce anti-CD36 alloimmune antibodies through the several pathways, such as blood transfusion, pregnancy or organ transplantation and so on, leading to the refractoriness of immune platelet transfusion and other diseases. The recent research advances of CD36 deficiency and its molecular biological basis, platelet transfusion and CD36 antibody detection are summarized briefey in this review.
Autoantibodies ; blood ; Blood Platelet Disorders ; Blood Platelets ; Blood Transfusion ; CD36 Antigens ; deficiency ; Female ; Genetic Diseases, Inborn ; Histocompatibility ; Humans ; Platelet Transfusion ; Pregnancy

OBJECTIVETo explore the molecular basis for a CD36 deficiency individual and distribution of CD36 gene mutation in Guangxi population.

METHODSA female individual was studied. CD36 phenotype was detected by monoclonal antibody immobilization of platelet antigens assay (MAIPA) and flow cytometry (FCM). The coding regions of the CD36 gene were sequenced. A DNA-based polymerase chain reaction-sequence specific primer (PCR-SSP) assay was used to verify the identified mutation. Cell lines expressing the mutant and wild-type CD36[CD36(MT) and CD36(WT)] were established, with the expression of CD36 determined by Western blotting. The distribution of CD36 gene mutation was investigated among 1010 unrelated individuals with the PCR-SSP assay.

RESULTSBoth MAIPA and FCM assays showed that the patient had type II CD36 deficiency. DNA sequencing showed that she has carried a heterozygous mutation T538C (Trp180Arg) in the exon 6 of CD36. Sequencing of cDNA clone confirmed that there was a nucleotide substitution at position 538 (538T>C). Western blotting also confirmed that the CD36 did not express on the CD36(MT) cell line that expressed the 538C mutant, but did express on the CD36(WT) cell line. The novel CD36 mutation T538C was further verified with 100% concordance of genotyping results by DNA-based PCR-SSP assay and 1010 unrelated individuals. No CD36 538C allele was detected among the 1010 individuals.

CONCLUSIONThis study has identified a novel CD36 mutation T538C(Trp180Arg)(GenBank: HM217022.1), and established a genotyping method for the novel sequence-specific primer PCR. The novel mutation is rare in Guangxi and can cause type II CD36 deficiency.

Base Sequence ; Blood Platelet Disorders ; genetics ; Blood Platelets ; cytology ; metabolism ; Blotting, Western ; CD36 Antigens ; genetics ; metabolism ; Cells, Cultured ; DNA Mutational Analysis ; DNA Primers ; genetics ; Exons ; genetics ; Female ; Flow Cytometry ; Fluorescent Antibody Technique ; Genetic Diseases, Inborn ; genetics ; Genotype ; Genotyping Techniques ; methods ; Humans ; Middle Aged ; Monocytes ; cytology ; metabolism ; Mutation, Missense ; Polymerase Chain Reaction ; methods

Inhibition of CD36, a fatty acid transporter, has been reported to prevent glucotoxicity and ameliorate high glucose induced beta cell dysfunction. Ezetimibe is a selective cholesterol absorption inhibitor that blocks Niemann Pick C1-like 1 protein, but may exert its effect through suppression of CD36. We attempted to clarify the beneficial effect of ezetimibe on insulin secreting cells and to determine whether this effect is related to change of CD36 expression. mRNA expression of insulin and CD36, intracellular peroxide level and glucose stimulated insulin secretion (GSIS) under normal (5.6 mM) or high glucose (30 mM) condition in INS-1 cells and primary rat islet cells were compared. Changes of the aforementioned factors with treatment with ezetimibe (20 μM) under normal or high glucose condition were also assessed. mRNA expression of insulin was decreased with high glucose, which was reversed by ezetimibe in both INS-1 cells and primary rat islets. CD36 mRNA expression was increased with high glucose, but decreased by ezetimibe in INS-1 cells and primary rat islets. Three-day treatment with high glucose resulted in an increase in intracellular peroxide level; however, it was decreased by treatment with ezetimibe. Decrease in GSIS by three-day treatment with high glucose was reversed by ezetimibe. Palmitate uptake following exposure to high glucose conditions for three days was significantly elevated, which was reversed by ezetimibe in INS-1 cells. Ezetimibe may prevent glucotoxicity in pancreatic β-cells through a decrease in fatty acid influx via inhibition of CD36.
Animals ; Anticholesteremic Agents/*pharmacology ; Antigens, CD36/antagonists & inhibitors/genetics/*metabolism ; Cells, Cultured ; Ezetimibe/*pharmacology ; Flow Cytometry ; Glucose/toxicity ; Insulin/genetics/metabolism/secretion ; Insulin-Secreting Cells/cytology/*drug effects/metabolism ; Male ; Palmitic Acid/metabolism ; RNA, Messenger/metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Real-Time Polymerase Chain Reaction