Effect and mechanism of tacrolimus on melanogenesis on A375 human melanoma cells

Background Topical tacrolimus has been used for vitiligo as a common treatment option for more than ten years while the underlying mechanism is still uncertain.The aim of this study was to investigate the direct effects of tacrolimus on the melanogenesis and migration on human A375 melanoma cells.The expression of c-KIT mRNA and protein of human A375 cells were also investigated.Methods The cultured A375 human melanoma cells were randomly assigned to control and tacrolimus treatment groups （10,102,103and 104 nmol/L）.The cell proliferation was measured with Cell Counting Kit-8 assays.Melanin content was measured with NaOH method.Transwell migration assay was used to measure cell migration.The expression of c-KIT mRNA and protein were measured with real-time fluorescence quantitative polymerase chain reaction and immunohistochemistry respectively.Results The cell proliferation of the 103 and 104 nmol/L tacrolimus groups were significantly lower （0.666±0.062 and 0.496±0.038） as compared with the control （0.841±0.110,P 〈0.05）.The mean melanin content in all groups treated with different concentration of tacrolimus （10,102,103,104 nmol/L） increased compared with the control group （P 〈0.05）.Dosedependent increase in cell migration were seen in all tacrolimus-treated groups （P 〈0.01）.The expression of c-KIT mRNA level in A375 cells exposed to tacrolimus （103and 104 nmol/L） had significantly increased by 3.03-fold and 3.19-fold respectively compared with the control （P 〈0.05）.Conclusions Although tacrolimus had no effects on cell proliferation on A375 human melanoma cells,it could increase the melanin content and cell migration.The expression of c-KIT mRNA and protein increased dose-dependently in tacrolimus-treated groups as compared with the control.Our study demonstrated that tacrolimus could enhance the melanogenesis and cell migration on A375 cells.     

The effect of spinal cord injury on the expression of TGF-β and TNF-α in rat articular cartilage

Objective: To observe the expression of TGF-β and TNF-α in the spinal cord injured rat model and discuss the significance of the articular cartilage metabolism. Methods: 36 SD female rats were randomly divided into 2 groups: Rats models of spinal cord injury were implemented by Allen method. T10 laminectomy was performed in the control group. Both groups of rats were killed respectively in 1w, 3w and 6w. Hematoxylin-eosin stain was given to each slice in the model group and control group. Immunohistochemical stain was applied by using ABC method in the expression of TGF-β and TNF-α. Those expressed level were performed in image analysis and statistics process. Results: TGF-β and TNF-α were mainly distributed on the surface layer of the articular cartilage, with a weak expression in control group. The expression of TNF-α in the model group was more significant than that in the control group in the 1w, and still remained an evident difference with that in control group until the 6w(P ＜ 0.05). TGF-β expression of the model group had no remarkable difference with the control group in the lw (P ＞ 0.05) and prominently became stronger at 6w(P ＜ 0.05). Conclusion: The expression of TNF-o occurred early in the development of spinal cord injury, and the expression of TGF-β became stronger with the revival of spinal neural function. Both expressions were strengthened in articular cartilage in the 3rd week.     

Effect of Coiquhoumia Root on the Expression of Transforming Growth Factor-β in Mesangiai Proliferation Giomerulonephritis Model

Summary： To study the efficacy and the mechanism of Colquhoumia root （ Tripterygium hypoglaucure （Le,vL） Hutch） in the treatment of mesangial proliferation glomerulonephritis （MsPGN）, SD rats were injected with anti-thymoeyte serum （ATS） to make MsPGN model （anti-Thyl model）. The rats were then divided into 3 groups： normal control group, anti-Thyl model group and treatment group. Histopathologieal （HE, PAS）, immunohistoehemieal, RT-PCR technique and computer imaging analysis system were used to evaluate mesangial matrix production, the expression of TGF-β protein and mRNA in the tissues of kidney. Our result showed that proteinuria and the ratio of extraeellular matrix/glomerular capillaries area （ECM/CA） were increased significantly in model group. The expression of both TGF-β protein and mRNA in glomeruli was much higher in model group than in control group （P〈0.01）. After the treatment with Colquhoumia root, proteinuria, ECM/CA and the expression of both TGF-β1 protein and mRNA in glomeruli were significantly decreased in treatment group as compared with those in model group. It is concluded that Colquhoumia root is effective in reducing proteinuria and mesangial matrix proliferation in MsPGN and it may achieve these effects by inhibiting the expressions of TGF-β1 protein and mRNA of mesangial cells.     

The effect of spinal cord injury on the expression of TGF-β and TNF-α in rat articular cartilage

Objective： To observe the expression of TGF-β and TNF-α in the spinal cord injured rat model and discuss the significance of the articular cartilage metabolism. Methods： 36 SD female rats were randomly divided into 2 groups： Rats models of spinal cord injury were implemented by Allen method. T10 laminectomy was performed in the control group. Both groups of rats were killed respectively in 1w, 3w and 6w. Hematoxylin-eosin stain was given to each slice in the model group and control group. Immunohistochemical stain was applied by using ABC method in the expression of TGF-β and TNF-α. Those expressed level were performed in image analysis and statistics process. Results： TGF-β and TNF-α were mainly distributed on the surface layer of the articular cartilage, with a weak expression in control group. The expression of TNF-α in the model group was more significant than that in the control group in the lw, and still remained an evident difference with that in control group until the 6w（P 〈 0.05）. TGF-β expression of the model group had no remarkable difference with the control group in the lw （P 〉 0.05） and prominently became stronger at 6w（P 〈 0.05）. Conclusion： The expression of TNF-α occurred early in the development of spinal cord injury, and the expression of TGF-β became stronger with the revival of spinal neural function. Both expressions were strengthened in articular cartilage in the 3rd week.     

Activation of nuclear factor κB and induction of inducible nitric oxide synthase by lipid-associated membrane proteins isolated from Mycoplasma penetrans

Background This study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans. Methods Mouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western blotting. The activity of nuclear factor KB (NF-κB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and Western blotting. Results M. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-κB activation, a possible mechanism for the induction of iNOS expression. Conclusion This study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-κB.     

Influence of all-trans retinoic acid(ATRA) on the expression of NANOG in Glioma cell lines

Objective To investigate the effects of all-trans retinoic acid(ATRA) on the expression of NANOG in glioma cell lines.Methods Each cell line was divided into the experimental group which was treated with ATRA for 5 days,and the control group which was cultured normally without ATRA treatment.Immunocytochemistry and RT-PCR were adopted to detect the expression of NANOG at protein and mRNA level among the three kinds of cell lines.Results Positive rates of NANOG protein in glioma cell lines SHG-44,U87 MG and U251 in control groups were(65.5±3.0)%,(64.8±8.0)% and(64.5±1.2)%,respectively,and the difference was not statistically significant(F=0.190,P=0.829).NANOG mRNA of the three cell lines in the relative content were 0.636 8±0.039 9,0.642 1±0.063 7,0.651 6±0.044 4,and the difference was not statistically significant(F=0.427,P=0.662).However,5 days after application of ATRA-induced NANOG protein in the three cell lines,the positive rates of NANOG protein of experimental groups were(36.5±7.3)%,(35.5±7.9)%,(35.2±6.1)%,respectively,compared with the control groups,the differences were statistically significant(FSHG-44=259.1,FU87=129.5,FU251= 431.8,PSHG-44=0.0,PU87=0.0,PU251=0.0),and the relative level of NANOG mRNA in these groups were 0.458 3±0.079 1,0.255 1±0.079 3 and 0.333 1±0.054 0,respectively,compared with the control groups,the difference was significant(FSHG-44=77.8,FU87=277.9,FU251=398.1,PSHG-44=0.0,PU87=0.0,PU251=0.0).Conclusion NANOG which highly expressed in glioma cell line SHG-44,U87 MG and U251 can be reduced by ATRA.     

生脉注射液对接受阿霉素治疗大鼠膈肌收缩力的作用

Objective:To explore the diaphragmatic toxicity in doxorubicin(DOX)-treated rats and the related mechanisms,as well as the effects of Shengmai Injection(SMI,生脉注射液)on the diaphragmatic dysfunction.Methods:Thirty Sprague-Dawley male rats were randomly divided into three groups:control,DOX-treated and DOX+SMI treated groups.DOX was given to rats in DOX and DOX+SMI groups in 6 equal doses[2.5 mg/kg,intraperitoneal injection(i.p.)],on alternate days,over a period of 2 weeks for a cumulative dose of 15 mg/kg.SMI was given to DOX+SMI rats in 12 doses(3 mL/kg,i.p.)for a period of 2 weeks before the administration of DOX and 2 weeks during the administration of DOX.The rats in the control group received equal volume of normal saline.Subsequently,the twitch and tetanic characteristics and force-frequency relationships,and the malondialdehyde(MDA)levels and the superoxide dismutase(SOD)activities,as well as the mRNA content and proteins of inducible nitric oxide synthase(iNOS)were determined.Results:The DOX-treated rats had decreased the peak twitch tension(Pt),maximal tetanic tension(P_0)and force-frequency relationship as compared with the control rats(P0.01),while the diaphragm contractility in rats treated with SMI were significantly higher than that in DOX-treated rats(P0.01).The DOX-treated rats had increased MAD levels and decreased SOD activities(P0.05),and SMI decreased the MDA levels and increased the SOD activities in DOX-treated rats(P0.05).Ultrastructure of diaphragm in the DOX-treated rats revealed typical alterations including fracture of diaphragm fibers,and edema and degeneration of mitochondria;these changes were relieved by SMI treatment.The mRNA content and protein of iNOS in DOX-treated rats were remarkably higher than those in control rats(P0.01),while SMI decreased the mRNA expression level of iNOS in DOX-treated rats(P0.05).Conclusions:Lipid peroxidation is responsible for DOX-induced diaphragm toxicity.SMI protects diaphragm muscles and their function from DOX impairment,and these beneficial effects may be somehow correlated with the decrease in expression of iNOS and lipid peroxidation.     

Effects of Nephritis No.3 Recipe on Nitric Oxide,Nitric Oxide Synthase Secreted by Cultured Mesangial Cells in Rats and the Gene Expression of Inducible Nitric Oxide Synthase

Objective:To explore the effect of the Nephritis No.3(N-3)recipe on nitric oxide(NO),nitric oxide synthase(NOS)secreted by cultured mesangial cells(MC)and its gene expression of the in-ducible nitric oxide synthase(iNOS).Methods:The drug(nephritis No.3)-containing serum was preparedwith serum pharmacological technique,and then was applied to react on mesangial cells cultured In fetalcalf serum(FCS)and cells cultured in FCS plus lipopolysaccharide.To observe the secretion of NO andNOS and the gene expression of iNOS by means of RT-PCR.Results:Under the two kinds of culture con-ditions,the content of NO and NOS in the groups with drug-containing serum were higher than thosewithout drug-containing serum(P<0.05,P<0.01),and the expression of iNOS mRNA was up-regula-ted too.Conclusion:The N-3 could significantly promote the secretion of NO and NOS and the mRNA ex-pression of iNOS in rats.     

Expression of NF-κB in hRPE Cells Stimulated by PDTC and IL-1β

The constitutive expression of nuclear-factor-κB (NF-κB) in human pigment epithelial (hRPE) cells cultivated in vitro and the possible changes when incubated with PDTC and IL-I were investigated. The synchronized hRPE cells in vitro were divided into two groups. In nonPDTC group, hRPE cells were exposed respectively to IL-1β and NS (for detecting the constitutive expressions of NF-κB in hRPE cells) ; In PDTC group, PDTC-pretreated hRPE cells were exposed respectively to IL-1β?Aand NS. (for detecting the constitutive expression of NF-κB in PDTC-pretreated hRPE cells). The expression of NF-κB in hRPE cells in two groups was detected by immunofluorescence stain and flow cytometry. The results showed that the constitutive expression of NF-κB in hRPE cells in vitro was 8.05 %, and increased to 30.26 % by IL-1β. After PDTC pretreatment, the constitutive expression of NF-κB in hRPE cells was decreased to 3.74%, and 3.66 % by IL-l,respectively. It was concluded that the expressions of NF-κB in hRPE cells could be increased significantly by IL-1βand depressed effectively by PDTC. Also, PDTC could significantly inhibit the activation of NF-κB induced by IL-1β.     

Effects of Needle-knife Therapy on iNOS Activity and NO Content in Skeletal Muscles in the L3 Transverse Process Syndrome Model Rat

Objective: To study on relationship of inducible nitric oxide synthase (iNOS) activity and nitric oxide (NO) content in the injured local soft tissue with injured degrees of the soft tissue in the third lumbar vertebrae (L3) transverse process syndrome model rat and to observe the effect of needle-knife therapy. Methods: One hundred and sixty male SD rats were randomly divided into normal group, model group, aminoguanidine (AG) group, needle-knife group, 40 rats in each group. The L3 transverse process syndrome rat model was established, and after treatment of needle-knife and AG, iNOS activities and NO contents and histomorpholocal changes in the soft tissues around L3 transverse process on 1, 3, 7 and 14 days were observed in the groups. Results: Compared with the normal group, iNOS activity and NO content in the model group were significantly increased (P<0.01); Compared with the model group, iNOS activities and NO contents were significantly decreased in both the needle-knife group and the AG group (both P<0.01); And both iNOS activities and NO contents were identical with both local inflammation response and injured degrees of the injured tissue in the groups. Conclusion: Needle-knife therapy can significantly inhibit generation of NO, alleviate inflammatory response and injured degree of the injured soft tissue, improve microcirculation, prevent formation of pathological scar tissue, and promote repair of the chronic soft tissue injury.     

Effect of Dexamethasone and Aquaporin-1 Antisense Oligonucleotides on the Aquaporin-1 Expression in Cultured Human Trabecular Meshwork Cells

The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.     

Effect of Chaiqin Chengqi Decoction(柴芩承气汤) on Cholecystokinin Receptor 1-Mediated Signal Transduction of Pancreatic Acinar Cells in Acute Necrotizing Pancreatitis Rats

Objective:To investigate the effect of Chaiqin Chengqi Decoction(柴芩承气汤,CQCQD)on cholecystokinin receptor 1(CCKRI)-mediated signal transduction of pancreatic acinar cell in rats with acute necrotic pancreatitis(ANP).Methods:Twenty-seven Sprague-Dawley rats were randomized into three groups:the control group,the ANP group,and the CQCQD group(9 in each group).ANP rats were induced by two intraperitoneal injections of 8%L-arginine(pH=7.0,4.4 g/kg) over a 2-h period.Rats were treated with 1.5 mL/100 g body weight of CQCQD(CQCQD group) or physiological saline(control and ANP groups) at 2 h interval.And 6 h after induction,pancreatic tissues were collected for histopathological examination.Pancreatic acinar cells were isolated for determination of CCKR1 mRNA and protein expression,phospholipase C(PLC) and inositol-1,4,5-triphosphate(IP3),and determination of fluorescence intensity(Fl)as a measure of intracellular calcium ion concentration[Ca~(2+)]_i.Results:The pancreatic histopathological score(6.2 + 1.1) and the levels of PLC(1,187.2 ±228.2 μg/mL) and IP3(872.2 ±88.4 μg/mL) of acinar cells in the ANP group were higher than those in the control(2.8 ±0.4,682.5 ±121.8 μg/mL,518.4 ±115.8 μg/mL)and the CQCQD(3.8 ±0.8,905.3 ±78.5 μg/mL,611.0 ±42.5 μg/mL) groups(P0.05).[Ca~(2+)]_i Fl for the ANP group(34.8 ±27.0) was higher than that in the control(5.1 ±2.2) and CQCQD(12.6 ±2.5) groups(P0.05).The expression of pancreatic acinar cell CCKR1 mRNA in the ANP group was up-regulated(expression ratio=1.761;P=0.024) compared with the control group.The expression of pancreatic acinar cell CCKR1 mRNA in the CQCQD group was down-regulated(expression ratio=0.311;P=0.035) compared with the ANP group.The ratio of gray values of the CCKR1 and B-actin in the ANP group(1.43 ±0.17) was higher than those in the control(0.70 ±0.15) and CQCQD(0.79 ±0.11) groups(P0.05).Conclusions:Pancreatic acinar cell calcium overload of ANP induced by L-arginine was related to the up-regulated expressions of pancreatic acinar cell CCKR1 mRNA and protein.CQCQD can down-regulate expressions of pancreatic acinar cell CCKR1 mRNA and protein to reduce the PLC and IP3 of pancreatic acinar cells,relieving the calcium overload and reducing the pathological changes in rats with ANP.     

Effects of aerosolized ketamine on the level of nitric oxide and nitric oxide synthetase in the lung tissue of rat with asthma

Objective： To explore the effects of aerosolized ketamine on the level of nitric oxide and nitric oxide synthetase in the lung tissue in rat asthma model. Methods： Forty SD rats were randomly assigned to five groups： control group （group N）, asthma model group （group A）, two pretreated groups of different concentrations of ketamine （group K1, K2） and dexamethasone group（group D） with eight rats in each group. The rats in group A were sensitized by injection of ovalbumin （OA） together with aluminum hydroxide and bordetella pertussis as adjuvants. Two weeks after the sensitization, aerosolized OA was used to cause asthma. The rats in group K1 and K2 were sensitized with OA as group A , and then exposed to aerosol of ketamine , with the concentration of 25 g/L and 50 g/L respectively. Before using aerosolized OA,the rats in group D were exposed to aerosol of 0.01% dexamethasone .The level of NO2^-/NO3^- in lung tissues, inducible nitric oxide synthetase（iNOS） and constitute nitric oxide synthetase（cNOS） was measured in all groups. Results： The level of NO2^-/NO3^- and the activity of iNOS in lung tissues in group A were signiticantly higher than those in the other groups. The iNOS activity and the level of NO2^-/NO3 ^- in lung tissues were highly positively correlated. Conclusion： NO can induce airway hyperreactivity that may worsen asthma. Aerosolized ketamine can decrease the iNOS expression and reduce the level of NO in the lung tissue in rat asthma model.     

Effects of Bushen Tiaochong Recipe (补肾调冲方) containing serum on ovarian granulosa cell proliferation, steroidogenesis and associated gene expression in rats

Objective:To observe the effect of Bushen Tiaochong Recipe (补肾调冲方,BSTCR) on rats' ovarian granulosa cell (GC) proliferation,steroidogenesis and follicle-stimulating hormone receptor (FSHR),and insulin-like growth factor-1 (IGF-1) mRNA expression using serum pharmacological method.Methods:Rats' GCs were incubated with 10% blank serum (as negative control group),follicle- stimulating hormone (FSH)-containing serum (S-FSH,as positive control group),or BSTCR (in different dosages) containing serum (S-BSTCR,as the BSTCR groups) for 48 h.~3H-TdR incorporation was then performed;DNA was measured to analyze the distribution of GCs in the cell cycle and their proliferation index (PI) using a flow cytometer;estradiol (E_2) and progesterone (P) content in the culture fluid were examined by radioimmunoassay;and levels of FSHR and IGF-1 mRNA expression in GCs were measured by real-time RT-PCR.Results:A dose-dependent increase of ~3H-TdR incorporation in GC was shown in the BSTCR groups.Cells in G_0/G_1 phase had markedly less,while those in S phase had a significantly higher increase in the BSTCR groups compared with the negative control group.A high value of PI was also shown in the BSTCR groups,especially in the high dose group where the influence of cell proliferation was stronger than that in the positive control group.The levels of E_2 and P in the BSTCR groups of all dosages were significantly higher than those in the negative control group,and did not show any significant difference compared with those in the positive control group.Levels of FSHR and IGF-1 mRNA expression in the BSTCR groups increased in a dose-dependent manner at levels higher than those in the negative control group.Conclusion:S-BSTCR can obviously stimulate the proliferation and steroidogenesis of ovarian GCs.It is speculated that BSTCR could play a regulatory action on ovarian function through two different pathways of endocrine and autocrine by promoting FSHR and IGF-1 mRNA expression.     

RNAi knockdown of C-erbB2 expression inhibits salivary gland adenoid cystic carcinoma SACC-83 cell growth in vitro

Objective：To knockdown the C-erbB2 gene in salivary gland adenoid cystic carcinoma SACC-83 cells using RNA interference,and determine the effect of silencing C-erbB2 on cell proliferation.Methods：C-erbB2-siRNA was transfected into SACC-83 cells.RT-PCR and immunohistochemistry were used to detect C-erbB2 expression in SACC-83 cells.Cell proliferation was measured by the MTT assay and gene knockdown was achieved by RNA interference.Apoptosis was analyzed by flow cytometry.Results：Compared with the control,C-erbB2 mRNA expression was decreased in the C-erbB2-siRNA transfection group,and immunohistochemical analysis indicated that C-erbB2 protein expression was decreased.After C-erbB2-siRNA was transfected for 48 h,absorbance at 570 nm （MTT）was 0.185±0.021 compared with 0.354±0.034,0.299±0.053,and 0.314±0.049 in the blank control,liposome control and negative control siRNA groups,respectively.The differences were statistically significant （P〈0.05）between the C-erbB2-siRNA group and the control groups.Following the C-erbB2 knockdown,the percentage of apoptotic cells was 5.63%compared with 2.04%,2.85%,and 2.98%in the three control groups,respectively.Proliferation of SACC-83 cells was inhibited,and early apoptotic cells were increased.Conclusion： RNA interference can effectively silence C-erbB2 gene expression and inhibit growth of SACC-83 cells,which indicates the potential of targeting this gene as a novel gene therapy approach for the treatment of salivary gland adenoid cystic carcinoma.