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Aim To explore the effects of niacin on LDL-C uptake and metabolism in HepG2 cells,and to clarify the functions of niacin in lipid-lowering and slo-wing the atherosclerosis process,thus to provide a sci-entific basis for niacin as a lipid-lowering drug in clini-cal development.Methods Oil red O staining was used to observe HepG2 cells after lipid uptake.Enzy-matic method was used to determine the content of in-tracellular free cholesterol (FC)and total cholesterol (TC).The LDLR levels on the surface of cell mem-brane were detected by immunofluorescence flow cy-tometer.The mRNA and protein expressions of LDLR, SREBP2 and PCSK9 were analyzed by qPCR and Western blot.Results The results of oil red O staining showed that the rate of oil red O-positive cells and the number of red lipid droplets were significantly in-creased in niacin group than control group.Niacin sig-nificantly increased the levels of TC and FC in HepG2 cells(P <0.05 ).What’s more,niacin significantly upregulated the expression of LDLR and significantly downregulated the protein expression of PCSK9,while it had no effect on the expression of SREBP2.Conclu-sion Niacin accelerates LDL-C uptake probably via downregulating the expression of PCSK9 and reducing the degradation of LDLR protein in HepG2 cells.
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Aim To explore the anti-proliferation effects of curcumin trinicotinate ( CurTn ) on vascular smooth muscle cell ( VSMC ) and its mechanism. Methods The cells were cultured in DMEM supple-mented with 10% fetal bovine serum. MTT assay was used to examine cell proliferation. FCM was used to observe cell cycle. The expressions of PCNA, Cy-clinD1 and p-ERK1/2 were analyzed using Western blot. Results CurTn could inhibit the proliferation of VSMC and showed a certain amount-time relationship. What’ s more, CurTn could increase the G1 phase pro-portion of cell, decrease the S phase proportion and the expression level of PCNA protein. It was also found that CurTn significantly inhibit the protein expression of p-ERK1/2 and Cyclin D1 . Conclusion CurTn may inhibit the proliferation of VSMC via downregulating the expression of CyclinD1 and p-ERK1/2 .
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Aim To explore the lipid-lowering mecha-nisms of curcumin from the molecular levels and pro-vide scientific basis for clinical development of lipid-lowering drugs.Methods Using oil red O staining and enzymic to determinate the levels of cholesterol in HepG2 cells.Moreover,uptaking of DiI-LDL was also measured.The expressions of mRNA and protein were detected by RT-Q-PCR and Western blot.Results The red lipid droplets and the levels of TC and FC sig-nificantly increased in HepG2 cells after treated with curcumin.The orange red fluorescence was higher than that of control.Curcumin could promote the expression levels of mRNA and protein of SREBP2 and LDLR, what′s more,curcumin could reduce the expression of the mature PCSK9 level and IDOL protein.Conclu-sion Curcumin accelerates LDL-C uptake probably via downregulating the expression of PCSK9 and IDOL in HepG2 cells.
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Objective To detect the relationship between the molecular defects and their phenotypes in children with growth hormone insensitivity syndrome (GHIS).Methods 21 patients defined as GHIS were enrolled in the study.4 candidate genes (GHR,IGFALS,JAK2,and STAT5B) were analyzed by genomic DNA sequence screening and clinical relevance analysis.Results The statistical descriptions of the patients were showed as an average height standard deviation (SDS)-4.33 ± 1.91 (-9.17 to-2.21),average serum peak values of GH (22.67 ±20.98) tg/L (11.33 to 104.21 μg/L),basal serum insulin-like growth factor-Ⅰ SDS-2.65 ± 0.53 (-3.57 to -1.79),insulin-like growth factor-binding protein 3 SDS-1.77 ± 1.64 (-4.13 to 0.96).Bone age of backward difference (chronological age-bone age) (43.10 ± 19.54) months (6 to 82 months).One of two children with severe growth failure and mid-face hypoplasia was found to a homozygote for G to A gene mutation in the intron 6 splice donor consensus sequences (IVS6 ds+ 1 G-A) in the GHR gene,causing its functional defect.3 cases with mild dwarf were found gene variations as novel finding:c.1097T＞C c.1098C＞T p.V366A pathogenic variant,c.1229C＞T p.S410L and nt1843707 A→G of 5' UTR region in the IGFALS gene.JAK2 and STAT5b genes mutations were not found.Conclusion Molecular pathology of GHIS is considered as involving the defects of GHR and its signal pathway.The mutation of intron 6 splice donor sequences in GHR gene has been reported which affect the function of GHR.The 3 novel type base variants in IGFALS gene,causing non severe dwarfism,might be suspected with pathogenic roles of GHIS.
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AIM:To investigate high-density lipoprotein ( HDL) subclass distribution and to analyze the rela-tionship between HDL subclasses with plasma glucose and lipids in metabolic syndrome ( MS) .METHODS:Apolipopro-tein 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 bio -chemical 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 HDL3b were increased in the MS patients (P<0.05).Meanwhile, HDL-C, apoA-I and preβ2-HDL, HDL2a and HDL2b were decreased in the MS patients (P<0.01).With the increase in the plasma glucose level , the contents of HDL2a and HDL2b 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 HDL2b 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 HDL2b had a decrea-sing 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 HDL2b, 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 HDL3b .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 me-tabolism of HDL particles is disrupted .
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Objective To compare the performance of 2％ (w/v) agarose gel,2％ (w/v) Metaphor agarose gel and 10％(w/v) nondenaturating polyacrylamide gel in the PCR-restriction fragment length polymorphism (RFLP) analysis of mycobacterial heat shock protein 65 (hsp65) gene.Methods This study included 8 Mycobacteria strains,including clinical isolates and standard strains of Mycobacteria tuberculosis and Mycobacterium intracellulare.Bacterialsuspension of these strains was prepared with the concentration of bacterial cells varying from 10 to 106per milliliter.PCR was performed to amplify the hsp65 gene with a pair of universal primers followed by the digestion of amplicons with two restriction endonucleases,BstE Ⅱ and Hae Ⅲ.Then,the restriction enzyme-digested fragments were subjected to electrophoresis in 2％ agarose gel,2％ Metaphor agarose gel and 10％ nondenaturating polyacrylamide gel respectively.Results As analysis of variance showed,the three gel electrophoresis methods were statistically different in sensitivity for the RFLP analysis of mycobacterial hsp65 gene (F =36.379,P ＜ 0.01).Least significance difference (LSD) procedure revealed that the 2％ agarose gel-based electrophoresis was less sensitive than the 2％ Metaphor agarose gel-and 10％ non-denaturing polyacrylamide gel-based electrophoresis (both P ＜ 0.01),and no significant differences were observed between the 2％ Metaphor agarose gel-and 10％ non-denaturing polyacrylamide gel-based electrophoresis (P ＞ 0.05).Conclusion The 2％ Metaphor agarose gel-and 10％nondenaturating polyacrylamide gel-based electrophoresis methods appear to be more sensitive than the 2％ agarose gel-based electrophoresis method for the PCR-RFLP analysis of mycobacterial hsp65 gene.
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Objective To evaluate the effect of the activity of Ca2+/CaN-NFATc on the activation and prolfferation of lymphocyte in asthmatic rats.Methods The rats of the asthma group and the CsA group were sensitized and challenged by ovalbumin.So did the control group with saline instead.Lymphocyte was separated from spleen and cultured for 24 hours,and PHA-p (5 μg/ml) was added to the culture medium in every group,CsA ( 1.0 μg/ml) was added to the CsA group,respectively.The concentration of [ Ca2+ ] i,the activity of CaN,the protein expression of dephosphorylated NFATc and Cyclin E in T lymphocyte were assayed.The level of IL-4 and IL-2 in culture supernatants was measured,and the cell cycle distribution of lymphocyte was analyzed.ResultsWhen compared to the control group,the activity of Ca2+/CaN-NFATc [ (81.21 4-14.39) vs (63.66 ±9.02) ] was increased and the protein expression of CyclinE [ (0.9327 ±0.0370) vs (0.8374 ±0.0637) ] was higher in Lymphocyte of the asthma group ( P＜0.05,P ＜0.01,respectively).The percentage of lymphocyte in the S phase [ (7.8600±2.8241) vs (4.0270 ± 1.8650) ] and S + G2/M phase [ ( 10.6700±3.3850) vs (5.8740 ± 1.4389) ] was higher;however,the percentage of G0/G1 phase [ (89.3300 ± 3.3850) vs (94.1260± 1.4389 )] was lower in asthma group ( all P ＜ 0.01 ).The level of IL-4 [ ( 1.55 ± 0.19) pg/ml vs (0.99 ± 0.12 ) pg/ml ] and the IL-4/ IL-2 ratio [ (0.81 ±0.12) vs (0.49 ±0.49) ] in culture supernatants of the asthma group were higher than those of the control group ( all P ＜0.01 ).While the activity of Ca2+/CaN-NFATc (47.19 ±7.16)was decreased and the protein expression of Cyclin E (0.6840 ± 0.0485 ) was reduced in lymphocyte in CsA group(all P ＜0.01 ),the percentage of lymphocyte in the S phase (4.8600 ± 1.9595) and S + G2/M phase (7.9900 ± 1.9405) was decreased and the percentage of G0/G1 phase (92.2100 ± 1.9267) was increased ( all P ＜ 0.05 ),and the level of IL-4 (0.47 ± 0.09 ) pg/ml and the ratio of IL-4/ IL-2 (0.78 ±0.20) was lower in culture supernatants in the CsA group than that of the asthma group( all P ＜ 0.01 ).There was a positive correlation between the protein expression of dephosphorylated NFATc and the protein expression of CyclinE in Lymphocyte,so did between the protein expression of NFATc and the level of IL-4 in culture supernatants( r =0.711,P ＜0.01 and r =0.749,P ＜0.01.respectively).Conclusions The activity of Ca2+/CaN-NFATc was increased in lymphocyte of the asthmatic rats.Its increasing might result in the imbalance of Th1/Th2 by promoting the expression of IL-4 and might lead to the proliferation of lymphocyte by promoting the Cyclin E expression.
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ObjectiveTo explore the influence of the cigarette smoke in the airway inflammation and pulmonary function of the asthmatic patients. MethodsTwenty-five cases of asthmatic patients with cigarette smoke exposure, 22 cases of asthmatic patients without cigarette smoke exposure and 20 cases of normal control persons were involved in this study. The proportion of various inflammatory cells in the induced sputum, the levels of serum interleukin (IL)-8 and IL-4 and lung function (FEV1％ expected value,FEV1/ＦVC％ ) were detected. ResultsThe infiltrating of neutrophils was primarily found in sputum of the asthmatic patients with cigarette smoke exposure, but the infiltrating of eosinophils was mainly in sputum of the asthmatic patients without cigarette smoke exposure. The levels of serum IL-8 and IL-4 of peripheral blood of asthmatic patients with cigarette smoke exposure[(277.02 ±71.37), (171.69 ±31.01) ng/L] were significantly higher than those in asthmatic patients without cigarette smoke exposure[(158.88 ± 21.95 ),( 111.42 ± 21.69 ) ng/L] and normal control persons [( 116.78 ± 71.37 ), (73.94 ± 15.72 ) ng/L] (P ＜ 0.01 ).The FEV1％ expected value and FEV1/FVC％ of the asthmatic patients with cigarette smoke exposure [(51.12 ± 13.30) ％, ( 49.16 ± 11.09 )％] was lower than those of asthmatic patients without cigarette smokeexposure [(81.81 ± 5.82)％, (79.00 ± 3.86)％] and normal control persona [(95.50 ± 10.11 )％, (83.18 ±6.04)％] (P ＜ 0.01 ). The level of serum IL-8 was positively correlated to the neutrophils percentage in the induced sputum (r =0.742,P＜ 0.01 ) ,while negatively correlated to the FEV1％ expected value(r =-0.739,P ＜ 0.01 ). ConclusionCigarette smoke may influence the airway inflammation of the asthmatic patients and accelerate the deterioration of their lung function by promoting the producing of IL-8.
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Objective To evaluate the activity of Ca2+ /CaN-NFATc, and study its association with the imbalance of TH1/ TH2 in asthmatic rat lungs. Methods Twenty-four Wistar rats were random divided to the asthma group and control group, twelve rats each group. The rats were sensitized and challenged with ovalbumin to establish the asthmatic model. The pulmonary function of rats was surveyed and evaluated u-sing Maclab system. Airway inflammation and the thickness of bronchial wall ( WAt/Pi) were observed by H. E staining. The quantity of Ca2+ , the activity of CaN , the protein expression of dephosphorylated NFATc and the level of IL-4 and IL-2 were assayed.Results Compared with control group, the thickness of bronchial wall was significantly increased ( t = -7. 99, P <0. 01), the airway resistance was higher( t = 2.59, P <0.05) and the respiration frequency was faster( t =7.94, P <0.01) ,but the minute ventilation volume was lower( t =6. 87, P <0.01) in asthma group. The levels of IL-4 and the IL-4/ IL-2 ratio in rat lungs of asthma group were significantly higher than those in control group ( t = -8.69, P <0. 01; t = 11.40, P <0. 01 .respectively) , however, the levels of IL-2 in asthma group were lower than that in control group ( t =8. 29, P <0. 01). The activity of CaN and the protein expression of dephosphorylated NFATc in asthma group were higher than those in control group( t = -2. 91, P <0.01; t = -22.45, P <0.01,respectively) ,but the quantity of Ca2+ in asthma group was lower than that in control group( t =4. 747, P < 0.01). There wag a positive correlation between the activity of CaN and the protein expression of dephos-phorylated NFATc( r =0. 39, P <0.05) ,so did between the protein expression of dephosphorylated NFATc and the IL-4/ IL-2 ratio( r =0. 83-, P <0.01) ,and the same between the thickness of bronchial wall and the IL-4/ IL-2 ratio( r = 0. 84, P < 0.01). Conclusions The activity of CaN-NFATc was increased in rat lungs of asthma group, and the rising of which might increase the ratio IL-4/ IL-2. Thus, the signal of CaN-NFATc probably took part in the imbalance of TH1/ TH2 in asthmatic rat lungs.
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Objective To observe the effects of intensive insulin therapy on C-reactive protein (CRP) ,interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α ) in the patients of critical illness complicated with hyperglycemia and its incidence of side effect. Methods Two hundred and nine patients of critical illness complicated with hyperglycemia were randomly divided into intensive insulin therapy group (106 patients,blood glucose maintained at a level of 4.4-6.1 mmol/L) and conventional insulin therapy group ( 103 patients, blood glucose maintained at a level of 9.0-11.1 mmol/L). Serum levels of CRP,TNF- α and IL-6 were determined on 0,24,48,72 h respectively after ICU admission. Results The levels of blood glucose of both groups reached the target level. The incidence rates of the hypoglycemia had no significant difference between two groups [6.60 % (7/106) vs. 4.76% ( 3/63 ),P > 0.05]. After 72 h treatment, serum level of CRP in intensive insulin therapy group was significantly lower than that in conventional insulin therapy group (P < 0.05 ). After 24,48 and 72 h treatment, serum level of IL-6 in intensive insulin therapy group was significantly lower than that in conventional insulin therapy group (P < 0.05 ). After 48 and 72 h treatment, serum level of TNF-αin intensive insulin therapy group was significantly lower than that in conventional insulin therapy group (P < 0.05). Conclusion Intensive insulin therapy can significantly decrease the levels of non-specific inflammatory factors in patients of critical illness complicated with hyperglycemia, which brings beneficial effect to the patients.
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Objective To test the drug susceptibility of 16 Mycobactena tuberculosis isolates from patients with cutaneous tuberculosis,and to provide a basis for the treatment of this entity.Methods Sixteen strains of mycobacterium were isolated from patients with cutaneous tuberculosis.and they were consistently identified as M.tuberculosis by biochemistry and molecular biology.Absolute concentration method was used to test the susceptibility of these isolates to isoniazid.streptomycin.rifampicin and ethambutol.For two strains resistant to streptomycin,PCR and sequencing were performed to analyse the mutation of rpsL gene.Results Out ofthe 16 strains of M tuberculosis.2 were resistant to streptomycin but sensitive to isoniazid,rifampicin and ethambutol,and 14 were sensitive to isoniazid,streptomycin,rifampicin and ethambutol.Sequencing of the rpsL gene revealed a mutation of AAG to AGG at codon 43 in one streptomycinresistant strain and a substitution of CGC bv CAC at codon 54 in another resistant strain.Conclusions In past five years,the resistance ratio of M tuberculosis was low in patients with cutaneous tuberculosis,and streptomycin resistance predominated in these strains.
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Objective To investigate the effect ofpeptidoglycan from Staphylococcus aureus on the release of several chemokines including intedeukin 8 (IL-8), regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage-derived chemokine (MDC) by normal human epidermal keratinocytes (KCs) and the role of Toll-like receptor 2 (TLR2) in this process. Methods KCs were derived from the foreskin of a healthy boy and propagated. After 2 - 4 passages, KCs were collected and treated with various concentrations (3, 10, 30 and 100 mg/L) of peptidoglycan for 24 hours or with peptidoglycan of 100 mg/L for varying durations (3, 6, 12, 36 hours). A fi'action of KCs were pretreated with functional grade purified anti-TLR2 monoclonal antibody before the treatment with peptidoglycan of 100 mg/L. After additional 12-hour culture following the treatment, enzyme linked immunosorbent assay was used to detect the level of IL-8, RANTES and MDC in culture supernatants of KCs. Results KCs spontaneously released IL-8 and RANTES. Peptidoglycan increased the production of IL-8 but decreased that of RANTES by KCs. The levels of IL-8 were 209.96 ± 10.31 ng/L, 250.28 ± 9.52 ng/L, 285.11 ± 10.28 ng/L, 359.40 ± 6.93 ng/L in KCs treated with peptidoglycan of 3, 10, 30, 100 mg/L, respectively, compared to 135.41 ± 14.37 ng/L in untreated KCs (all P < 0.05). On the contrary, a significant decrement was seen in the secretion of RANTES by KCs treated with peptidoglycan of 10, 30, 100 mg/L compared with untreated KCs (110.72 ± 8.51 ng/L, 90.50 ±2.45 ng/L, 49.89 ± 13.74 ng/L vs 149.94 ± 18.71 ng/L, all P < 0.05). The monoclonal antibody to TLR-2 could markedly suppress the promotion of IL-8 production by peptidoglycan, but had no obvious influence on the inhibition of RANTES production by peptidoglycan. MDC could not be detected in the culture super-natants of KCs with or without peptidoglycan stimulation. Conclusion Peptidoglycan could inhibit RANTES secretion but induce IL-8 production by KCs likely via TLR2.
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Objective To study the effect of soluble, refolded, recombinant extracellular domain of the human Fc gamma receptor Ⅱ a (huFcγRⅡa) on the binding of human IgG to cells. Methods Extra-cellular domain of the huFcγRⅡ a gene was amplified from recombinant plasmid pe3huR Ⅱ by PCR and then cloned into pET-28a vector. The recombinant plasmid pETshuR Ⅱ was transformed into E. coli BL21 (DE3) after identified by PCR and doubly digested. The inclusion bodies of fusion protein were extracted and purified by washing, dissolved in 6 mol/L guanidine buffer, and refolded by rapid dilution technique. The refolding protein activity was tested by ELISA and flow cytometry. Results Extraceilular domain of the huFcγRⅡa gene was successfully cloned into pET-28a. The results of SDS-PAGE showed that the molecular mass (Mr) of the expressed protein was 24.8 × 103, and the expression rate was 30%. The purity of recom-binant shuR Ⅱ was up to 90% after washing. ELISA showed that the recombinant shuR Ⅱ was able to bind human IgG in a dose dependent manner, shuRⅡ could competitively inhibit the binding of human IgG to huFcγRⅡa expressed on the surface of COS-7 cells by flow cytometry. Conclusion The results demon-strate that it is possible to obtain large quantities of recombinant shuR Ⅱ which has comparable binding prep-erties to those of the whole membrane bound huFcγR Ⅱ a.
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AIM:To observe the expression of cyclin E in the lungs of asthmatic rats and to evaluate the role of cyclin E in airway remodeling with asthma. METHODS:Twenty Wistar rats were randomized to the asthma group and control group (10 rats in each group). The rats were sensitized and challenged with ovalbumin to establish the asthmatic model. Airway inflammation was observed by HE staining. The thickness of the bronchial wall (WA/Pi) was measured by computer-assisted image analysis system. The cell cycle distribution of monocytes in peripheral blood was analyzed by flow cytometry. The mRNA expression of cyclin E was detected by realtime RT-PCR. The protein expression of cyclin E was assayed by Western blotting and immunohistochemistry. RESULTS:The thickness of the bronchial wall in asthma group was significantly higher than that in control group (P