Effect of yigu capsule drug-containing serum on proliferation,ALP activity and expression of interleukin-11 mRNA in rat osteoblasts in the co- culture

AIM:To investigate the effect of yigu caps ule drug-containing serum on proliferation,ALP activity and expression of inter leukin-11 mRNA in Sprague-Dawley rat osteobalsts in the co-culture system.METHODS:(1) Osteoblasts and osteoclasts were isolated from 1 an d 5-day-old Sprague-Dawley rats,respectively.The osteoblasts-osteoclasts co- culture system was built to prevent the two kinds of cells from contact and allo w the media to exchange.The experiment included two groups,drug-containing se ra group and control group.(2) The osteoblasts proliferation,ALP activity and expression of interleukin-11 mRNA were detected by the MTT,4-aminoantipyrine sp ectrometric methods and FQ-PCR，respectively.RESULTS:In drug-containing sera group,the osteoblasts prolifer ation,ALP activity,expression of IL-11 mRNA were higher than those of control group (P<0.05).CONCLUSION:Rat sera containing yigu capsule can obviously enhan ce the osteoblast proliferation,ALP activity and expression of IL-11 mRNA.     

Effect of benefiting-bone Capsule on IL-6 mRNA and protein expression in rat osteoblasts in vitro

AIM: The effects of benefiting-bone Capsule (BBC) containing serum on IL-6 mRNA and protein expression in osteoblasts were studied.METHODS: (1) The neonate Sprague-dawley rat osteoblasts were cultured and divided into three groups: group Ⅰ (containing deactivating serum with BBC), group Ⅱ (containing deactivating serum without BBC) and group Ⅲ (DMEM medium group)； (2) RT-PCR was used to measure the relative IL-6 mRNA levels； (3) The radioimmunoassay method was used to examine IL-6 protein in the supernatant of the cultured osteoblasts. RESULTS: (1) The relative IL-6 mRNA levels was lower in group I than the control (P<0.05); (2) The IL-6 protein expression in osteoblasts was also lower in group I than the control (P<0.05).CONCLUSIONS: These results suggest BBC drug-containing serum can down-regulate the expression of IL-6 mRNA and protein in osteoblasts, which may be one of the mechanisms of BBC preventing and treating osteoporosis.     

Effect of TNF-α and Yigu capsule drug-containing sera on osteoblasts apoptosis in coculture system

AIM: To observe the effect of TNF-α and Yigu capsule drug-containing sera on osteoblasts apoptosis in osteoblasts-osteoclasts coculture system.METHODS: (1) Twenty female Sprague-Dawley rats, 10 months old, were randomly assigned into 2 groups, NS and Yigu capsule groups, to prepare blank sera and drug-containing sera. (2) The DNA gel electrophoresis method was used to detect apoptosis of osteoblasts treated with different concentration of TNF-α in order to determine the best dosage in the co-culture system. (3) The cells were divided into four groups, TNF-α group, normal group, TNF-α + blank serum group and TNF-α + drug-containing serum group. DNA gel electrophoresis, acridine orange staining and flow cytometry were used to observe osteoblast apoptosis in these groups. RESULTS: (1) After induced by TNF-α for 72 h at a concentration of 60 μg/L, relatively typical DNA ladder appeared in TNF-α group. (2) Only two DNA brands appeared and most of cells were well-proportioned stained in TNF-α + drug-containing serum group, the rate of osteoblasts apoptosis in TNF-α + drug-containing serum group (9.60%±0.26%) was obviously lower than that in the TNF-α group (26.90%±0.06%) and TNF-α + blank serum group (18.10%±0.06%). CONCLUSION: TNF-α induces osteoblast apoptosis in the co-culture system, and Yigu capsule drug- containing serum prevents osteoblast apoptosis induced by TNF-α.     

Effect of Xiaoyaosan on proliferation and differentiation of hippocampus nerve precursor cells under high corticosterone condition

AIM:To investigate the effect of Xiaoyaosan (XYS) drug-containing serum on proliferation and differentiation of hippocampus nerve precursor cells (HNPC) under high corticosterone (CORT) concentration condition. METHODS:Serum-free culture in vitro was adopted to culture HNPC. CORT at concentration of 120 μmol/L was used to establish the high CORT concentration condition. XYS is composed of Bupleurum Chinese DC, Chinese angelica root, Paeonia lactiflora Pal1, India bread, Largehead atractylodes rhizome, peppermint herb, fresh ginger and Licorice root. XYS drug-containing serum of different dose were prepared.10% drug-containing serum and 10% serum were adopted. RU38486, an antagonist of CORT, was used as positive control. MTT method was used to detect proliferation rate of HNPC, the methods of immunofluorescence ambi-tagged by BrdU with TUNEL, β-tubulin-Ⅲ and glial fibrillary acidic protein (GFAP) with TUNEL respectively were adopted to detect the proliferation and differentiation of HNPC. RESULTS:CORT at concentration of 120 μmol/L degraded the proliferation rate of HNPC significantly (P<0.01), which was reversed by RU38486 (P<0.05). 10% XYS drug-containing serum of each dose enhanced the proliferation rate (P<0.05 or P<0.01). After 120 μmol/L CORT processed, the staining fluorescence intensity ratio of BrdU/TUNEL of HNPC degraded significantly (P<0.01), while increased after treatment with RU38486 and 10% XYS drug-containing serum of each dose (P<0.01). Under high CORT concentration condition, the apoptotic rates of glial cells and neurons differentiated from HNPC increased significantly (P<0.01). RU38486 and 10% XYS drug-containing serum of each dose inhibited the apoptotic rates of glial cells and neurons (P<0.05 or P<0.01). CONCLUSION:Under high CORT concentration condition, XYS promotes the proliferation of HNPC, and inhibits the apoptotic rates of glial cells and neurons differentiated from HNPC, which act as the effect of anti-damage of CORT to hippocampus in stress state.     

Effect of Yigu capsule drug-containing serum on apoptosis and tartrate resistant acid phosphatase secretion in rat osteoclasts

AIM:The effects of Yigu capsule on tartrate resistant acid phosphatase (TRAP) secretion and apoptosis in rat osteoclasts were investigated in order to further explore its mechanism of preventing and treating osteoporosis.METHODS:(1) Twenty-month-old Sprague-daweley rats were randomly divided into two groups(Yigu capsule group and saline group), and the drug-containing serum and control serum were prepared. (2) The newborn Sprague-daweley rat osteoclasts were cultured with different concentrations of Yigu capsule drug-containing serum. At different time point, TRAP activity was measured and the survival osteoclast was counted under reverse microscope.The percentage of osteoclast apoptosis was observed under fluorescence microscope after acridine orange staining.RESULTS:TRAP activity was lower and the percentage of osteoclast apoptosis was higher in drug-containing serum group than in control group at 24, 48 and 72 h(P<0.01), respectively, and the survival osteoclasts were less in drug-containing serum group than in control groups at 24, 48 and 72 h(P<0.01).CONCLUSIONS:These data suggest that Yigu capsule drug-containing serum induces apoptosis and inhibits TRAP activity in osteoclasts, which may be one of the mechanisms of Yigu capsule preventing and treating osteoporosis.     

Effects of insulin on osteoblast and its post-receptor mechanism

AIM:To study the effects of insulin on the proliferation and function of osteoblasts and the relationship between insulin post-receptor change in osteoblasts and osteoblastic cell growth.METHODS:The effects of different levels of insulin on osteoblasts were assessed by MTT colorimetry.Osteocalcin in medium was measured by RIM.IGF-1 mRNA expression levels were determined by RT-PCR.The concentrations of free IGF-1 protein in serum-free medium were measured by ELISA.In addition,the protein level and phosphorylated protein of P44/42MAPK were determined by Western blotting analysis.RESULTS:Insulin enhanced the proliferation of osteoblasts,depending on its dose and exposure time.Insulin at concentration of 10-7 mol/L showed the strongest effect,and the action attained the plateau phase beyond 96 h.The best concentration that stimulated synthesis of osteocalcin by insulin was 10-7 mol/L.When the insulin concentration beyond 10-7 mol/L,the osteocalcin concentration was decreased.Exposure time had no effect on insulin-stimulated synthesis of osteocalcin of osteoblastic cells.When the concentration of insulin reaches 10-6 mol/L,the IGF-1 mRNA expression stimulated by insulin was also decreased.The concentrations of free IGF-1 protein in insulin-stimulated groups were all higher than that in control group (P<0.05),but there was no statistically significant difference among insulin-stimulated groups (P>0.05).Insulin acute stimulation rapidly induced the activity of tyrosine phosphorylation of P44/42MAPK.The degree of tyrosine phosphorylation of P44/42MAPK was increased step by step along with the increasing doses of insulin from 0 to 10-7 mol/L (P<0.05,between groups).After insulin chronicity treatment,there was a marked reduction in the tyrosine phosphorylation of P44/42 MAPK (P<0.05,between groups).There was no significant change in protein level of P44/42MAPK.CONCLUSIONS:Insulin enhances the proliferation of osteoblasts as a growth factor at a suitable concentration,but this effect disappears at chronic high insulin stimulation.The MAPK may be involved in the proliferating effect of insulin on osteoblasts.Transient stimulation of insulin activates the P44/42MAPK,however chronic high insulin stimulation results in down-regulation of P44/42MAPK signal activity.     

Effects of DEHP on testosterone synthesis in fetal Leydig cells of newborn male rats

AIM: To observe the effects of diethylhexylphthalate(DEHP) on testosterone synthesis in fetal Leydig cells(FLC) of newborn male rats.METHODS: The pregnant rats were exposed to DEHP at dose of 10 mg·kg^-1·d^-1, 100 mg·kg^-1·d^-1 or 750 mg·kg^-1·d^-1(body weight) by gavage from gestation 12 days(GD 12) to postnatal 1 day(PND 1) respectively. The serum level of testosterone was detected by chemiluminescence. The morphology of FLC from the testes was observed under light microscope and transmission electron microscope. The mRNA expression of steroidogenic acute regulatory protein(StAR) and insulin-like growth factor I(IGF-I) was detected by real-time PCR(ΔΔCT). RESULTS: The serum testosterone level in low dose group was significantly higher than that in control, middle and high dose groups. The serum testosterone level in middle and high dose groups was significantly lower than that in control group(P<0.05). Light microscopy showed the aggregative cluster distribution of FLCs in low dose group, while manifested as tumor-like hyperplasia of FLCs in middile dose group and high dose group. Under electron microscope, the FLC in low dose group showed oval-shape or long spindle-shape, the lipid particles were decreased, but smooth endoplasmic reticulum and mitochondria were increased in cytoplasm. In middle and high dose group, the FLC were spindle or oval-shaped, showed large or small, nuclear large, round, rich in cytoplasm and cell aggregation, the cytoplasm was rich lipid particles with deep-stained, smooth endoplasmic reticulum and mitochondria were expanded. Compared to control group, the mRNA expression of StAR in low, middle and high dose group was decreased. The mRNA expression of StAR in high dose group was decreased more significantly(P<0.01) as compared to that in control group, while the mRNA expression of IGF-I in low dose group and middle dose group was increased, but that only in low dose group was increased more significantly as compared to control group(P<0.01). CONCLUSION: DEHP has toxic effect on FLC and changes the morphology and steroidogenic capacity in testicular FLC. The low dose of DEHP elevates the gene expression of IGF-I, and IGF-I stimulates the production of testosterone by FLC. The high dose of DEHP may inhibit the gene expression of StAR to reduce the serum levels of testosterone.     

Effect of drug-containing serum of Liuwei Dihuang pills on TGF-β/Smad signaling pathway in HK-2 cells

AIM: To investigate the effect of the drug-containing serum of Liuwei Dihuang pills on the TGF-β₁/Smad signaling pathway in HK-2 cells.METHODS: The proliferation of HK-2 cell was detected by MTT method. Western blotting analysis was used to investigate the effect of the drug-containing serum of Liuwei Dihuang pills on the protein expression of the molecules of Smad signal transduction pathway.RESULTS: The drug-containing serum of Liuwei Dihuang pills promoted the proliferation of HK-2 cells. The level of Smad2 phosphorylation in HK-2 cells treated with 10% drug-containing serum of Liuwei Dihuang pills was significantly lower than that in the cells treated with TGF-β₁. Furthermore, SnoN, a negative factor in Smad signaling pathway, was up-regulated in HK-2 cells treated with 10% drug-containing serum.CONCLUSION: Drug-containing serum of Liuwei Dihuang pills inhibits TGF-β₁/Smad signaling pathway, including reducing Smad2 phosphorylation and promoting SnoN protein expression.     

Effects of Gax gene transfection on proto-oncogene expression and proliferation of PASMCs in rats under hypoxia conditions

AIM: To explore the effects of Gax gene transfection on expressions of c-fos and c-jun mRNA and proliferation of pulmonary arterial smooth muscle cells (PASMCs) in rat under hypoxia.METHODS: PASMCs were transfected with Gax gene by Ad-Gax.Under normal oxygentention (21% O₂) or hypoxia (2.5% O₂) for 12 h condition,expressions of Gax mRNA and protein in PASMCs were detected by RT-PCR and immunocytochemistry.The expressions of c-fos and c-jun mRNA were evaluated by RT-PCR.［³H］-TdR incorporation was used to measure the PASMCs proliferation.RESULTS: The Gax overexpression in transfection group was confirmed by RT-PCR and immunocytochemistry.Under normal oxygentention or hypoxia,the c-fos and c-jun mRNA levels in transfection group were lower than those in the non-transfection group,respectively (P<0.05).［³H］-TdR incorporation in the transfection group was lower than that in non-transfection group (P<0.05,P<0.01).CONCLUSION: Gax overexpression might inhibit the PASMCs proliferation induced by hypoxia through downregulating the expressions of c-fos and c-jun.     

Effect of combination of Penthorum chinense Pursh and Puerariae flos on L-02 liver cell injury induced by alcohol

AIM: To explore the effect of Penthorum chinense Pursh and Puerariae flos-containing serum on L-02 liver cell injury induced by alcohol and its possible mechanism. METHODS: After preparing drug-containing serum, the L-02 cells cultured in vitro were divided into 6 groups:blank control group, model group, 1:1 group, 2:1 group and 1:2 group of combination of Penthorum chinense Pursh and Puerariae flos, and tiopronin group. The viability of the L-02 cells was measured by MTT assay. The activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and superoxide dismutase (SOD), and the content of malondialdehyde (MDA) were detected by enzyme label methods. The expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) at mRNA and protein levels was determined by real-time PCR and Western blot, respectively. RESULTS: Compared with control group, the levels of ALT, AST and MDA were increased significantly, and SOD was decreased in model group (P<0.01). Compared with model group, these indexes in all treatment groups were opposite (P<0.01). Compared with control group, the expression of TNF-α and IL-6 at mRNA and protein levels was significantly increased, the mRNA and protein expression of Nrf2 and HO-1 was decreased (P<0.01). Compared with model group, these indexes in combination groups were opposite (P<0.01). CONCLUSION: The therapeutic effects of Pentehorum chinensa Pursh and Puerariae flos-containing serum may affect the expression levels of TNF-α, IL-6, Nrf2 and HO-1, and reduce the inflammatory reaction and oxidative stress in alcohol-induced L-02 liver cells, which plays a role in attenuating alcoholic liver injury.     

Effect of Tripterygium hypoglaucum Hutch on MMP-13 protein expression and IL-12, IL-23 and IL-37 levels in serum and foot paws of rats with collagen-induced arthritis

AIM: To investigate the effect of tripterygium hypoglaucum Hutch (THH) on collagen-induced arthritis (CIA) in rats and its possible mechanism. METHODS: SD rats were randomly divided into normal group and CIA group. The rat model of type Ⅱ CIA was successfully established and the model rats were randomly divided into 4 different groups: model group, dexamethasone group, THH (200 mg/kg) group, and THH (400 mg/kg) group. The contents of IL-12, IL-23 and IL-37 in the serum and foot paws of the CIA rats were detected by ELISA. The histopathological changes of the skin of the food paws were observed by HE staining. The protein expression of MMP-13 was determined by Western blot. The MMP-13 activity in the foot paws was detected by fluorescence labeling method. RESULTS: Compared with CIA group, THH at dose of 400 mg/kg significantly reduced the weight loss in type Ⅱ CIA rats (P<0.01). THH at dose of 400 mg/kg obviously decreased the contents of IL-12 by 28.31%, IL-23 by 41.57% in the serum and IL-12 by 30.78%, IL-23 by 39.46% in the foot paws, while IL-37 was significantly increased by 79.43% in the serum and 75.78% in the foot paws (P<0.01). The pathological changes of the subcutaneous tissues were improved by treating with THH (400 mg/kg). The protein expression of MMP-13 was significantly decreased by 31.82% (P<0.01), and the MMP-13 activity was also reduced. THH at dose of 200 mg/kg had no obvious improvement on the above indexes. CONCLUSION: THH has significant inhibitory effect on rat CIA by reducing the content of proinflammatory cytokines IL-12 and IL-23, increasing the content of anti-inflammatory factor IL-37, inhibiting inflammatory cell infiltration and vascular proliferation, and attenuating the protein expression of MMP-13 and MMP-13 activity in rats.     

mRNA expression of insulin receptor and leptin receptor in liver of nonalcoholic fatty liver disease from diabetic rats

AIM: To observe the pathologic changes of liver in diabetic rats and to investigate the role of mRNA expression of insulin receptor and leptin receptor in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). METHODS: Twenty male Sprague-Dawley rats were divided randomly into two groups: normal control group and diabetic group. After fed with high-fat diet for 4 weeks, diabetic rats were injected with streptozotocin at a dosage of 30 mg/kg intraperitoneally to induce NAFLD model of type 2 diabetes mellitus. Then the diabetic animals were fed with high-fat diet continuously for 12 weeks. At the end of the experiment, the rats were sacrificed, the concentrations of blood glucose, serum lipid, ALT and AST were measured biochemically. The levels of serum leptin and serum insulin were detected by enzyme-linked immunosorbent assay (ELISA) and radio immunoassay (RIA), respectively. The pathologic changes of liver were observed under light microscopy (LM) stained with HE, Sudan Ⅲ and Masson trichrome staining, respectively. The ultra-structural changes of liver were observed under transmission electron microscopy (TEM). Additionally, the mRNA expressions of PEPCK, G6Pase, insulin R and leptin R from rat livers were assayed by semi-quantitative RT-PCR. RESULTS: The levels of blood glucose, serum insulin, serum TG, ALT and AST increased significantly (P<0.01), serum TC elevated (P<0.05), and the levels of serum leptin decreased (P<0.01) in diabetic group compared to those in normal control group. Obvious liver fatty degeneration, piecemeal necrosis with accompanying inflammatory infiltration and fibrosis were found under LM. Hepatocytes pyknosis, lots of lipid deposits in cytoplasm of hepatocytes, proliferation of collagen in space of Disse were observed under TEM in diabetic group. The expression of insulin R and leptin R mRNA in liver from diabetic rats increased significantly (P<0.01) while the expression of PEPCK and G6Pase mRNA remained unchanged. CONCLUSION: Insulin resistance plays an important role in the pathogenesis of NAFLD. Low level of serum leptin, up-regulation of mRNA expression of insulin R and leptin R in liver caused by insulin resistance may be involved in this process.     

Effects of long-term cigarette smoke exposure on pulmonary vascular remodeling and TGF-β1 expression in rat pulmonary vessels

AIM: To observe the effects of long-term cigarette smoke exposure on pulmonary vascular remodeling and the protein expression of transforming growth factor-β1(TGF-β1) in the rats, and to explore the mechanism.METHODS: SD rats(n=36) were randomly divided into control group, 2-week smoke exposure(S-2W) group and 12-week smoke exposure(S-12W) groups. HE staining and α-smooth muscle actin staining were performed to observe the pulmonary vascular remodeling.The protein expression of proliferating cell nuclear antigen(PCNA) and TGF-β1 in the pulmonary arteries was determined by the method of immunohistochemistry. The mRNA expression of TGF-β1 in the pulmonary arteries was evaluated by RT-qPCR.RESULTS: Compared with control group, ratio of pulmonary vessel wall thickness to vessel diameter(WT%) and percentage of muscularized vessels were significantly increased in S-2W group and S-12W group(P<0.01). Significant increases in the protein expression of PCNA and TGF-β1 in smoke exposure groups were observed compared with control group. There was significant difference between 2 model groups(P<0.01). Compared with control group, the mRNA expression of TGF-β1 in pulmonary artery walls obviously increased in smoke exposure groups. There was significantly difference between S-2W and S-12W groups(P<0.05). The mRNA expression of TGF-β1 was positively correlated with pulmonary vascular muscularization, WT% and the protein expression of PCNA. CONCLUSION: Long-term cigarette smoke exposure results in pulmonary artery remodeling in rats. The possible mechanism is that cigarette smoking exposure induces the over-expression of TGF-β1 at mRNA level in pulmonary vessels and promotes the proliferation of pulmonary vascular smooth muscle cells in rats.     

Effects of puerarin on apelin-12, angiotensin II,NO and blood pressure in renovascular hypertensive rat

AIM: To investigate the effects of puerarin on blood pressure in renovascular hypertensive rats, and to measure puerarin-induced changes of apelin-12, angiotensin II (Ang II) and nitric oxide(NO), the factors related to development of hypertension. METHODS: Sixty-five male Sprague-Dawley rats were used, of which 8 rats were randomly selected as sham operation group, and the remaining were used to make two-kidney, one-clip model. The rats that met the criterion for Goldblatt hypertensive rat model were randomly allocated into 5 groups: high-, middle- and low-dose puerarin groups, captopril group, and model group. The drugs were administered for 6 weeks. Blood pressure was measured every 2 weeks. Six weeks after treatment, all rats were sacrificed under deep anesthesia. Blood and kidney samples were collected. The level of apelin-12 in serum and kidneys was detected by ELISA. The level of Ang II in plasma and kidneys was measured by radioimmunoassay. NO level in serum was examined by nitrate reductase assay. RESULTS: Puerarin had an antihypertensive effect in a dose-dependent manner. Marked decreases in the level of serum apelin-12 in high- and middle-dose puerarin groups were observed(P<0.01). Puerarin at low dose did not cause obvious change in the content of apelin but still reached significant level (P<0.05). As the dose of puerarin went up, the level of apelin-12 in the kidneys was gradually decreased. Puerarin at high and middle doses obviously reduced the level of AngII in plasma, while purarin at low dose did not produce any significant effects. Puerarin at high and middle doses markedly increased the level of NO in serum, but puerarin at low dose did not induce any significant changes. CONCLUSION: Puerarin has an antihypertensive effect, and its mechanism may be related to inducing the changes of apelin, Ang II and NO, and regulating the balance among those factors.     

Protective effects of luteolin on STZ-induced diabetic kidneys

AIM: To explore the protective effects of luteolin on the diabetic kidneys. METHODS: Male Sprague-Dawley rats were randomly divided into 5 groups: normal control group, diabetic model group and the groups of diabetic rats treated with luteolin at a low dose, a middle dose and a high dose. The diabetic model was induced by a single intraperitoneal injection of streptozotocin (STZ,65 mg/kg). Blood glucose, urine protein, the activity of superoxide dismutase and catalase in serum and kidney, and the content of malonaldehyde(MDA) in kidney were analyzed by biochemical methods. Western blotting was used to detect the protein expression of transforming growth factor-β₁(TGF-β₁) and plasminogen activator inhibitor-1(PAI-1) in the renal cortex. The morphological changes of the renal tissues were observed under microscope. RESULTS: Compared with diabetic model group, luteolin significantly reduced the level of blood glucose (P<0.01), the content of urine protein (P<0.01) and MDA (P<0.01) in the kidneys, and increased the activity of superoxide dismutase and catalase (P<0.01) in serum and kidneys in the diabetic rats. The protein levels of TGF-β₁ and PAI-1 in the renal cortex were dramatically decreased as the rats were treated with luteolin. CONCLUSION: Luteolin may exert an important protective effect on diabetic kidneys by relieving oxidative stress and inhibiting the protein expression of TGF-β₁ and PAI-1 in the renal tissues of STZ-induced diabetic rats.     

Effect of 308 nm excimer laser on expression and secretion of basic fibroblast growth factor and cell viability in human keratinocytes

AIM: To investigate the effect of 308 nm excimer laser on the cell viability, and mRNA expression and protein secretion of basic fibroblast growth factor(bFGF) in human keratinocytes(hKCs).METHODS: Human keratinocytes cultured in vitro were identified by immunofluorescence method and radiated with 308 nm excimer laser and 311 nm narrow-band ultraviolet B(NB-UVB) at different dosages, respectively. MTT method was used to measure the cell viability. The mRNA expression and protein secretion of bFGF were detected by the methods of RT-PCR and ELISA,respectively. The cells treated with NB-UVB were used for comparison. RESULTS: The cytoplasm of the cells presented kelly fluorescence under the fluorescent microscope, indicating that the cells were keratin positive. The cell viability were decreased by the exposure to 600 mj/cm 2. of 308 nm excimer laser or 500 mj/cm 2. of NB-UVB. The inhibitory effect of 308 nm excimer laser on the cell viability was slighter than that of NB-UVB. In both 308 nm excimer laser group and NB-UVB group, the mRNA expression and protein secretion of bFGF were significantly higher than those in control group and increased in a dose-dependent manner. The cells in 308 nm excimer laser groups had higher secretion level of bFGF than that in NB-UVB groups in certain range of dosages. CONCLUSION: The effect of 308 nm excimer laser on the cell viability is better than that of NB-UVB. The upregulation of bFGF production in keratinocytes by 308 nm excimer laser is in a dose-dependent manner, so as to promote the repigmentation of vitiligo lesions.     

Expression of OPN and macrophage colony stimulating factor in diabetic rats and intervention of mycophenolate mofetil

AIM: To explore the effect of mycophenolate mofetil (MMF) on expression of osteopotin (OPN) and macrophage colony stimulating factor (M-CSF) in diabetic rats with renal tubulo-interstitial injury. METHODS: Diabetes was induced in uninephrectomized male Wistar rats by peritoneal injection of streptozotocin (65 mg/kg). The rats were randomly divided into three groups: control group (NC), diabetic group (DM) and MMF treated group ［DM+MMF, treatment of MMF (15 mg·kg^-1·d^-1) by gavage from the next day of the induction for 8 weeks］. Serum biochemistry, 24 h urinary protein and the ratio of left kidney weight/body weight were determined after 8 weeks. The renal tubulo-interstitial morphological change was observed, immunohistochemical method was used to analyze the expression of OPN, M-CSF and CD68. The mRNA of OPN in renal tissue was amplified by quantitative real-time PCR. RESULTS: Compared with control group, serum glucose level, 24 h urinary protein and the ratio of left kidney weight/body weight were significantly increased (P<0.01), and the relative area of interstitial fibrosis was also significantly enlarged in DM group (P<0.01).Compared with NC group, the expressions of OPN, M-CSF, CD68 protein and OPN mRNA were significantly upregulated in DM group (P<0.01). After intervention with MMF, the upregulations of the above-mentioned parameters, except blood glucose and serum creatinine, were all significantly inhibited (P<0.05 or P<0.01). CONCLUSION: The expressions of OPN, M-CSF and CD68 in renal tubulointerstitial decrease in diabetic rats treated with MMF. MMF also inhibits the level of OPN mRNA, reduces proteinuria and prevents renal injury. MMF plays an apparently protective role in renal tubulointerstitial injury, probably associated with inhibiting chemokine and proliferation on macrophages.     

Effect of TRPC6 on PDGF-induced proliferation of rat airway smooth muscle cells

AIM:To investigate the role of canonical transient receptor potential channel 6 (TRPC6) in the proliferation of airway smooth muscle cells (ASMCs) induced by platelet-derived growth factor (PDGF). METHODS:Rat ASMCs were cultured by enzyme digestion and tissue adhesion. The method of indirect immunofluorescence was applied to identify the ASMCs and to detect the expression of TRPC6 in ASMCs. The proliferation of ASMCs was determined by CCK-8 assay. The mRNA expression of TRPC6 was tested by real-time PCR. The protein expression of TRPC6 was analyzed by Western blotting. The influence of TRPC6 blocker at different concentrations on the proliferation of ASMCs was measured by CCK-8 assay. RESULTS:The results of immunofluorescence indicated that TRPC6 expression in ASMCs was positive. PDGF at concentration of 20 μg/L induced the proliferation of ASMCs compared with control group (P<0.05). When ASMCs were treated with both PDGF and different concentrations of TRPC6 blocker SKF96365, the proliferation of ASMCs was attenuated in dose-dependent and time-dependent manners as compared with the cells treated with PDGF alone (P<0.05). The mRNA expression of TRPC6 in PDGF group was significantly increased (P<0.05) after ASMCs were treated with PDGF for 12 h, 24 h and 48 h. The protein level of TRPC6 in PDGF group was significantly increased after ASMCs were treated with PDGF for 24 h and 48 h compared with control group (P<0.05). CONCLUSION: Up-regulation of TRPC6 at mRNA and protein levels is most possibly related to the proliferation of ASMCs induced by PDGF. Therefore, TRPC6 is involved in the proliferation of ASMCs.     

Effects of perindopril at different doses on cardiac function and ACE2/Ang-(1-9)/Ang-(1-7) axis of ischemic cardiac dysfunction rabbits

AIM: To investigate the different dose of perindopril on cardiac function in the rabbits with ischemic cardiac dysfunction. METHODS: Male rabbits weighing 2.5~3.0 kg(n=30) were randomly divided into 3 groups(n=10):high dose perindopril group(HD group), low dose perindopril group(LD group) and cardiac dysfunction group(CD group). The Left anterior descending coronary artery of the rabbits was ligatured for model preparation. In HD group, the rabbits were treated with perindopril split normal saline solution(1 g/L)2 mL·kg^-1·d^-1. In LD group, the rabbits were treated with perindopril split normal saline solution(0.33 g/L)2 mL·kg^-1·d^-1. In CD group, the rabbits were treated with normal saline solution 2 mL·kg^-1·d^-1. Four weeks after treatment, the cardiac function was measured via echocardiography, the mRNA expression of angiotensin-converting enzyme 2(ACE2) and angiotensin type 2 receptor(AT2R) was analyzed by real-time PCR, serum angiotensin(Ang)-(1-9) and Ang-(1-7) levels were detected by ELISA. RESULTS: Compared with CD group, the cardiac function of the 2 groups treated with perindopril was significantly improved(P<0.01), and more improvement in HD group was observed than LD group(P<0.05). The serum angiotensin(Ang)-(1-9) and Ang-(1-7) level and the mRNA expression of ACE2 and AT2R in the 2 groups treated with perindopril were significantly improved(P<0.01). Compared with LD group, the mRNA expression of ACE2 and AT2R and the serum levels of Ang-(1-9) in HD group were significant improved(P<0.05), while no difference of serum Ang-(1-7) level was observed. Correlation analysis revealed that the improvement of the cardiac function was associated with serum Ang-(1-9) level, mRNA expression of ACE2 and AT2R(P<0.01), but has no significant correlation with serum Ang-(1-7) level. CONCLUSION: High dose of perindopril may improve more cardiac function in ischemic cardiac dysfunction model in rabbits. The mechanism may relate to increasing serum Ang-(1-7) level to activate AT2R.