Effect of Yangyu Tuji on content of type Ⅰ, Ⅲ collagen and the expression of MMPs and TIMP-1 in wound caused by streptozotocin in rats

AIM: To study the effects of Yangyu Tuji (YYTJ) on delayed healing wound of diabetic rats caused by streptozotocin (STZ). METHODS: SD male rats were randomly divided into control group (control), model group (model); and 3 different dose groups of YYTJ. 55 mg/kg STZ were given by intraperitoneal injection except for control group. After 30 days, a round skin of 1.6 cm diametre was excised on all dorsal back of rats. The healing time and healing rate were observed according to re-epithelization. The content of collagenⅠ and Ⅲ was observed by Picric acid-Sirius red staining , Matrix metalloproteinase-1, 13 (MMP-1, -13), tissue inhibitor of metalloproteinases-1 (TIMP-1) by immuno-histochemistry assay. All data were analyzed by IPP software. RESULTS: The healing time in each group treated with YYTJ was shorter than that in model group (P<0.01), and the healing rate was increased (P<0.01, P<0.05). Content of type I collagen, ratio of type Ⅰ and Ⅲ collagen of high and mid dose group were significantly higher than that in model group (P<0.01) at 3rd, 7th, 11th day. The expression of MMP-1, -13 of each groups were higher than that in model group at 7th day (P<0.01, P<0.05), and MMP-1 trend to equal with model group at 11th day. MMP-13 was significantly lower than that in model group at 11th day (P<0.01, P<0.05). TIMP-1 of each group of wound was higher than that in model group at 3rd, 7th, 11th day (P<0.01, P<0.05). The ratio of type Ⅰ and Ⅲ collagens in each group was lower than that in model group at 11th day (P<0.01). Ratio of MMP-13 and TIMP-1 of high dose group and mid dose group were higher than that in model group at 3rd and 7th day (P<0.01). The ratio of each group was lower than that in model group at 11th day (P<0.01). Meanwhile, ratio of MMP-13 and TIMP-1 of high dose group and mid dose group were lower than that of lower dose group (P<0.05). CONCLUSION: It is possible that YYTJ accelerates wound healing by increasing collagen content of type Ⅰ and Ⅲ, especially type Ⅰ, as well as improves collagen deposition by regulating the balance of MMP and TIMP.     

Effect of recombinant MIF on lung fibroblast proliferation and collagen synthesis in vitro

AIM: To investigate the influence and mechanism of recombinant macrophage migration inhibitory factor (rMIF) on fibroblasts. METHODS: MRC-5 fibroblasts were divided into two groups: the treated group was treated with rMIF (25-100 μg/L, 12 h, 24 h or 48 h) and the control was non-rMIF treatment. The activity of proliferation in both groups was investigated and compared by CCK-8 means. Synthesis of collagen in the culture supernatants was detected by the hydroxyproline. The expression of collagen type I mRNA was examined using RT-PCR analysis. The level of collagen type I protein induced by rMIF was quantified by Western blotting. RESULTS: The production of proliferation ratio of fibroblasts treated with 50 μg/L and 100 μg/L rMIF at 24 h or 48 h were increased obviously (P<0.05 or P<0.01). The collagen synthesis significantly increased after stimulation with 100 μg/L rMIF for 48 h (P<0.01). rMIF significantly increased the expression of collagen type I mRNA and protein in a dose dependent manner compared with control (P<0.05 or P<0.01). CONCLUSION: rMIF upregulates the proliferation of fibroblasts and has an effect on the production of collagen. These results suggest that MIF may play important roles in the pathogenesis of airway remodeling.     

Protective effects of Zhenwu decoction on kidney injury of diabetic rats induced by streptozotocin

AIM：To investigate the effects of Zhenwu decoction (ZWD) on the expression of α-smooth muscle actin (α-SMA) and NF-κB in diabetic nephropathy (DN) rats. METHODS：Diabetic rat model was induced by intrape-ritoneal injection of streptozotocin (STZ), and the animals were randomly divided into STZ group (n=22) and STZ+ZWD group (n=23). The normal rats served as control (n=16). All rats were sacrificed on 8 weeks after modeling. Biochemical assay and pathological observation (HE staining and transmission electron microscopy) were used to evaluate the effects of Zhenwu decoction on the renal function and pathological morphology. The body weight, renal index, blood glucose, total urinary protein in 24 h, and superoxide dismutase (SOD), malondialdehyde (MDA),inducible nitric oxide synthase (iNOS) were determined as well. Western blotting was used to observe the effects of Zhenwu decoction on the expression of α-SMA and NF-κB in diabetic nephropathy (DN) rats. RESULTS：Compared with normal group, the renal index, blood glucose concentration, total urinary protein in 24 h, blood urea nitrogen (BUN), serum creatinine (SCr) and MDA were significantly higher and body weight was lower in DN rats (P<0. 05). Pathological examination of the kidneys in DN group showed glomerular hypertrophy, glomerular basement membrane thickening, tubular epithelial cell degeneration, mesangial matrix proliferation, protein cast formation in some renal tubules. The protein expression levels of α-SMA and NF-κB were markedly increased (P<0.05). After ZWD treatment, the level of renal index, total urinary protein in 24 h, BUN, SCr and the expression of α-SMA and NF-κB at the protein level were significantly decreased (P<0.05). The renal histological injury in ZWD group was significantly ameliorated. CONCLUSION：Zhenwu decoction might protect kidney against STZ-induced injury via decreasing the expression of α-SMA and NF-κB.     

Effect of acarbose on the aortic collagen nonenzymatic glycosylation in diabetic rats

AIM: To observe the effect of α-glucosindase inhibitor, acarbose on the aortic collagen nonenzymatic glycosylation in streptozotocin-induced diabetic rats . METHODS: The treated group (DM+A) was given acarbose (1 mg·kg^-1). The aortic collagen and its AGEs concentration were measured at the scheduled periods (1, 3 and 6 months ). RESULTS: During the observed period , the aortic collagen and its AGEs concentration were higher than that of control group in a time-dependent manner (P＜0.01 ), but decreased in acarbose-treated rats as compared with untreated group (P＜0.01 ). CONCLUTION:Acarbose can prevent the aortic vascular collagen nonenzymatic glycosylation in diabetic rats.     

Effects of basic fibroblast growth factor on proliferation and collagen production in human umbilical cord mesenchymal stem cells

AIM：To observe the growth pattern and surface markers of human umbilical cord mesenchymal stem cells(hUCMSCs) and to explore the influence of basic fibroblast growth factor (bFGF) on the proliferation and collagen production in hUCMSCs cultured in vitro. METHODS：hUCMSCs were isolated by enzyme digestion method and adherent culture. The surface markers CD45, CD34, CD105, CD29 and HLA-DR of the cells were analyzed by flow cytometry. The osteogenic ability and adipogenic differentiation were confirmed with oil red O and alizarin red staining. The optimal concentration of bFGF to promote the proliferation of hUCMSCs was 20 μg/L. The cells in control group were cultured in the growth medium consisting of DMEM/F12 and 10% volume fraction of fetal bovine serum. The cells in experiment group were cultured under the same condition of control group but plus 20 μg/L bFGF. The proliferation of hUCMSCs was analyzed by MTT assay. The expression of type I and III collagens at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS：The growth curves indicated that bFGF promoted the proliferation of hUCMSCs. The hUCMSCs expressed CD29, but did not express CD34, CD45 or HLA-DR in the presence or absence of bFGF unanimously. The cells were alizarin red staining-positive and oil red O staining-positive. Compared with control group, the expression of type I and III collagens significantly decreased at mRNA and protein levels in experiment group. CONCLUSION：bFGF promotes the proliferation of hUCMSCs and does not change the expression of the surface markers. bFGF inhibits the expression of type I and III collagens at mRNA and protein levels, indicating that bFGF enhances the healing of wound without inducing scar hyperplasia.     

Effects of hydrogen sulfide on wound healing of skin ulcer in diabetic rats

AIM: To study the effect of hydrogen sulfide on wound healing of skin ulcer in diabetic rats.METHODS: Male SD rats were randomly divided into 3 groups, including non-diabetic control(NDC) group, untreated diabetic control(UDC) group, and treated diabetic administration(TDA) group. Diabetic rats were induced by intraperitoneal injection of streptozotocin(STZ). After 1 week, wound healing model was prepared by making a round incision(2.0 cm in diameter) on the dorsal skin in full thickness. The rats from TDA group received 2% sodium bisulfide ointment on the skin ulcer wound, and the animals from UDC and NDC groups received control cream. After 21 d of treatment with sodium bisulfide, blood samples were collected for biochemical analysis, including prothrombin time(PT), thrombin time(TT), and fibrinogen(FIB) in plasma, as well as the activity of superoxide dismutase(SOD) and the content of malondialdehyde(MDA) in the serum. White blood cells(WBC) and lymphocytes were also counted. Granulation tissues from the wound were processed for histological examination and Western blot analysis was used to detect heme oxygenase-1(HO-1) and tumor necrosis factor α(TNF-α) expression.RESULTS: Compared with UDC group, sodium bisulfide treatment accelerated wound healing of skin ulcer(P<0.01), and increased the activity of SOD in serum(P<0.01) in the diabetic rats. The declined number of WBC and lymphocytes, prolonged PT and TT, and decreased FIB levels in rats treated with sodium bisulfied were also confirmed. Pathological section showed that there were inflammatory cell infiltration, and irregular and loose fibril alignment in the granulation tissue of rats from the UDC group, but there were regular fibril alignment and increased angiogenesis in the granulation tissue of rats from the TDA group(P<0.05). Furthermore, sodium bisulfide treatment raised HO-1 protein expression, and decreased TNF-α protein expression in the diabetic rats.CONCLUSION: Hydrogen sulfide accelerates the wound healing of skin ulcer in the rats with diabetes. The beneficial effect of H₂S may be associated with formation of granulation, anti-inflammation, and antioxidation.     

Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases during wound healing in diabetic rats

AIM: To investigate the imbalance between the expression of metalloproteinases (MMPs) and that of tissue inhibitors of metalloproteinase (TIMPs) during wound healing in diabetic rats. METHODS: Diabetic rats were induced with streptozotocin. All rats were maintained for 6 weeks. A full-thickness excisional wound was created on the back of each rat. Every group was randomly divided into 3 subgroups of 7 rats: 3 d group, 7 d group, 14 d group and animals were killed at 3rd, 7th and 14th day. Routine pathological examination, Masson′s trichrome staining and immunohistochemistry were made to calculate the score of epidermal and dermal regeneration, granulation tissue thickness, angiogenesis, matrix density, and infiltrated cells at different time points. RT-PCR and Western blotting were used to detect the expression of mRNA and protein of MMP-9 and TIMP-1 in the skin at those time points. RESULTS: Six weeks after streptozotocin treatment, Three days after injury, the wound healing rate of normal rats was faster than that of diabetic rats. From 3rd to 14th day, there were a lot of fibroblast and macrophage in normal skin, while few such cells were observed in diabetic skin. The other histological scores in normal skin were higher than those in diabetic rats at 7th and 14th day. Both MMP-9 and TIMP-1 had minimally detectable levels before wounding but exhibited rapid, significantly large increases within 3 d after wounding. Subsequently, they showed a rapid decline by 14 d. The relative values of expression of MMP-9 mRNA and protein in diabetic group were higher than those in normal group at different time points. However, the values of TIMP-1 mRNA and protein in diabetic group were significantly lower than those in control group. Significant difference was observed between two groups with the ratio of MMP-9/TIMP-1, higher in diabetic group than that in normal group. CONCLUSION: Abnormal reepithelialization, angiogenesis, inflammatory cell infiltration, collagen fibers generation, granulation tissue deposition, seem to be the basic histopathology that delays wound healing. The imbalance between MMPs and TIMPs in diabetic skin tissue before and after injury may be one of the important reasons of these alterations of histopathology.     

Effect of fibronectin on proliferation and collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats

AIM:To investigate the effects of fibronectin (FN) on the proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFb) derived from SHR (CFb_SHR) and WKY (CFb_WKY). METHODS:CFb derived from 12-week-old spontaneously hypertensive rat (SHR) and WKY was cultured by outgrowth of tissue block. Cell proliferation of CFb was measured by cell number counting and[³H]-TdR incorporation using 24-well plates pre-coated with 5 μg/cm² of FN. Collagen synthesis was determined by [³H]-proline incorporation. RESULTS:As compared with control, the cell number of fibroblasts derived from SHR and WKY were significantly increased to 163.75% and 170.42% respectively after 72 h incubation with FN in the presence of 0.4% FCS from a intial cell density of 1×10⁴ cells/mL. DNA synthesis of CFb was markedly promoted by FN. FN induced an increased in [³H]-proline incorporation in both CFb_SHR and CFb_WKY. CONCLUSION:FN is able to promote cell proliferation and collagen synthesis of CFb derived both from SHR and WKY.     

Roles of Rho-kinase in rat cardiac fibroblasts proliferation and collagen synthesis induced by angiotensin Ⅱ

AIM: To investigate the role of Rho-kinase signal pathway in rat cardiac fibroblasts (CFBs) proliferation and collagen synthesis induced by angiotensinⅡ (AngⅡ). METHODS: CFBs of neonatal Sprague-Dawley (SD) rats were isolated with the method of trypsin digestion and differential anchoring velocity. The CFBs were stimulated with AngⅡto induce fibrosis. Proliferation of CFBs was observed by MTT coloricmetric assay. Synthesis of collagen was detected by the hydroxyproline. The expression of Rho-kinase mRNA was examined using RT-PCR analysis. The extent of phosphorylation of myosin-binding subunit (MBS-P) of myosin phosphatase was quantified by Western blotting analysis, which was used to evaluate the activity of Rho-kinase.RESULTS: (1) Stimulation of neonatal SD rat CFBs with AngⅡ (10-7 mol/L) significantly increased CFBs proliferation and collagen synthesis (P<0.01). (2）Stimulation of neonatal SD rat CFBs with AngⅡ (10-7 mol/L) significantly increased the expression of Rho-kinase mRNA and rapidly activated Rho-kinase in a time-dependent manner. (3) Within a concentration coverage, hydroxyfasudil (H4413), a Rho-kinase inhibitor, effectively inhibited AngⅡ-induced CFBs proliferation and collagen synthesis (P<0.05 or P<0.01).CONCLUSION: Rho-kinase signal pathway may be one of the most important signal transducter for AngⅡ-induced CFBs proliferation and collagen synthesis in neonatal SD rats.     

Effects of insulin on cardiac fibroblast proliferation and cardiac myocyte hypertrophy

AIM:To study the effect of insulin on proliferation and hypertrophy of cardiac myocytes and its role in the induction of cardiac hypertrophy. METHODS:1. The neonatal rat cardiac myocytes and cardiac fibroblasts were cultured respectively and identified with light microscopy, electron microscopy and immunocytochemistry. 2. Cell proliferation was measured with cell number, metabolic activity and DNA synthesis (with WST-1, BrdU enzyme-linked immunosorbent assay ) and the percentage of S+G₂+M in cell cycle (by flow cytometry ). 3.Cell hypertrophy was evaluated by cell protein content (Coomassie Briliant Blue's method). RESULTS:1. The cultured cells showed the characteristic of cardiac myocytes and cardiac fibroblasts, respectively. 2. After being treated with insulin, the cell number, absorbance of BrdU incorporation and WST-1 cleavage products and the percentage of S+G₂+M of cardiac fibroblasts increased significantly (P＜0.01 orP＜0.05), while the above parameters of cardiac myocytes remained unchanged (P＞0.05). 3. Protein content of cardiac myocytes increased significantly in a dose-dependent manner (P＜0.01 orP＜0.05) in insulin treated groups (10^-10 mol/L-10^-7 mol/L). CONCLUSION:Insulin promoted cardiac fibroblast proliferation and increased myocytes protein content(induced myocyte hypertrophy)in vitroand may play an important role in pathogenesis of cardiac hypertrophyin vivo.     

Apoptosis in diabetic foot ulcers and human fibroblast cells treated with AGEs

AIM：To investigate cell apoptosis in diabetic foot ulcers and the effect of advanced glycosylation end products (AGEs) on apoptosis in human fibroblast cells. METHODS：Diabetic foot patients (n=18) and 18 age-matched non-diabetic controls were recruited. The clinical and biochemical features were compared by statistics methods. Skin biopsies were obtained from foot. Cleaved caspase-3 was measured by immunohistochemistry using the technique of streptavidin-biotin complex (SABC) staining. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique was used to detect apoptosis of the skin tissues. Human primary foreskin fibroblasts were isolated and cultured in the presence of 5.6 mmo/L glucose, 25 mmo/L glucose, fluctuant glucose (changing the glucose from 5.6 mmo/L to 25 mmo/L every 8 h) or AGEs (150 mg/L, containing 5.6 mmo/L glucose). After 72 h treatment, Western blotting was used to determine the levels of the apoptotic protein cleaved-caspase-3. Other cells were trypsinized, washed with cold PBS and incubated with PI and Annexin V-FITC, then analyzed by flow cytometry to detect cell apoptosis. RESULTS：Diabetic patients had higher levels of fasting blood glucose (FBG), 2-hour postprandial blood glucose (2 h PBG) and glycosylated hemoglobin A1c (HbA1c), and longer wound duration. The protein level of cleaved caspase-3 was significantly higher in diabetic group, suggesting that apoptosis was increased in diabetic skin tissues. TUNEL analysis showed that apoptotic index was higher in diabetic group compared with that in non-diabetic group (8.4%±1.5% vs 3.8%±08%), which further confirmed that cell apoptosis was increased in diabetic foot tissues. In human fibroblasts, the levels of cleaved caspase-3 in normal group, sustained high glucose group, fluctuant high glucose group and AGEs group were 080±0.13, 1.22±0.18, 1.46±0.32 and 1.83±0.25, respectively. The apoptotic rates detected by flow cytometry were 2.43%±0.19%, 2.89%±0.51%, 3.99%±0.24% and 6.83%±0.36%, respectively. Both the level of cleaved caspase-3 and the apoptotic rate in AGEs group were higher than those in normal glucose group and sustained high glucose group. CONCLUSION：Increased apoptosis in diabetic foot ulcers is one of the most important reasons for impaired wound healing. As compared to sustained high glucose and glucose fluctuations, AGEs induce greater apoptosis in human fibroblast cells.     

Effects of Ginkgo biloba extract on myocardial TGF-β1 and collagen expression and interstitial fibrosis in type I diabetic cardiomyopathy rats

AIM:To study the effects of Ginkgo biloba extract (EGB) on myocardial TGF-β1 and collagen expression and interstitial fibrosis in type I diabetic cardiomyopathy rats. METHODS:Thirty male SD rats were randomly divided into normal control group (CON), diabetes mellitus group (DM) and EGB treatment group (EGB). Streptozocin was intraperitoneally injected into the animals in the latter 2 groups to induce type I diabetic rat model. The rats in EGB group were intraperitoneally injected with EGB. At the end of the 12th week, the body weight of each rat and its left ventri-cular weight, blood glucose, glycosylated hemoglobin and serum insulin concentration were measured. The left ventricular end-diastolic volume (LVEDV), the left ventricular end-systolic volume (LVESV), the left ventricular ejection fraction (LVEF) and the stroke volume (SV) were determined by echocardiography. The content of collagen in left ventricular myocardium, and the expression of transforming growth factor β1 (TGF-β1), procollagen type I and collagen type III were assayed by Sirius red staining, immunohistochemical staining and RT-PCR, respectively. Left ventricular myocardial cells of the neonatal SD rats were isolated and cultured in vitro with low-glucose culture medium (LG group), high-glucose culture medium (HG group) or high-glucose culture medium plus EGB (HG+EGB group). The mRNA levels of TGF-β1, procollagen type I and collagen type III were detected by RT-PCR. RESULTS:Compared with CON group, blood glucose, glycosylated hemoglobin, left ventricular weight index, the content of collagen, and the expression of TGF-β1, procollagen type I and collagen type III in left ventricular myocardial tissues of DM group were significantly increased, while the levels of blood insulin, LVEDV and SV were significantly decreased. However, compared with DM group, blood glucose, glycosylated hemoglobin, left ventricule weight index, the content of collagen, and the expression levels of TGF-β1, procollagen type I and collagen type III in the left ventricular myocardial tissues of EGB-treated rats were significantly decreased, while the levels of blood insulin, LVEDV and SV were significantly increased. Compared with LG group, the mRNA expression levels of TGF-β1, procollagen type I and collagen type III were significantly increased. However, compared with HG group, the mRNA expression levels of TGF-β1, procollagen type I and collagen type III were significantly decreased after treated with EGB. CONCLUSION: EGB retards the process of myocardial fibrosis and improves the cardiac functions in type I diabetic cardiomyopathy rats by down-regulating the expression of TGF-β1, reducing the synthesis and deposition of collagen type I and collagen type III.     

Intracerebroventricular injection of streptozotocin induces dysfunction of insulin signaling in brain and cognitive deficits in rats

AIM：To investigate the effect of insulin signaling on the development of Alzheimer disease (AD) and to explore its potential mechanisms. METHODS：The rats were treated with streptozotocin (STZ, 3 mg/kg) intracerebroventricularly (ICV) at the 1st day and the 3rd day of the experiment to induce dementia model. Twenty-one days after the injection of STZ at the 1st day, spatial learning and memory of the rats were determined by Morris water maze test. The expression levels of insulin-degrading enzyme (IDE), glycogen synthase kinase 3β (GSK-3β), phosphorylated GSK-3β (p-GSK-3β), tau and phosphorylated tau (p-tau) were measured by Western blotting. The levels of amyloid β-proteins (Aβ1-40 and Aβ1-42) in the brain of the rats were detected by the method of immunohistochemistry. The mRNA levels of insulin, insulin receptor, tau and IDE were measured by real-time RT-PCR. RESULTS：ICV-STZ deteriorated the abilities of spatial learning and memory of the rats and reduced the activity of IDE and the mRNA levels of insulin and insulin receptor. STZ treatment enhanced GSK-3β activity and tau phosphorylation. The levels of Aβ1-40 and Aβ1-42 in cerebral cortex were significantly increased in the rats treated with STZ. CONCLUSION：ICV-STZ results in AD-like behaviors and pathological changes via damaging the brain insulin signaling, indicating that insulin signaling may play important roles in the AD pathogenesis.     

Role of stress in the lung injury caused by acute hemorrhagic necrotizing pancreatitis in rats

AIM:To study the relation ship between stress and lung injury caused by acute hemorrhagic necrotizing pancreatitis(AHNP) through AHNP model.METHODS:The AHNP model was made by using 5% sodium taurocholate retrograded injection into biliopancreatic duct in SD rats. Those rats were divided into three groups randomly, from A to C, the A group undertook sham operations, the B group was made into AHNP model, and the C group was given Metyrapone. The level of corticosteroid, CRP and amylase in serum had been observed. The lung and pancreas histological examinations were also performed.RESULTS: In C group, the level of corticosteroid, CRP and amylase were much lower than those in B group. The grade of lung and pancreas injury were also lower than those in B group(P＜0.05).CONCLUSION:Stress lays an important role in the lung injury caused by AHNP. Using anti-stress drugs can inhibit this course and meliorate the lung injury.     

Basic fibroblast growth factor promoting the healing of palatal perforation in rats

AIM: To study the influence of basic fibroblast growth factor(bFGF) on treating ed palatal perforation in rats. METHODS: bFGF was given to the early palatal perforation in rat. The granulation tissues in perforations were grossly and pathologically obserVed. RESULTS: The the of wound healing was significantly in- crease in the bFGF group (P<0.01 ). bFGF could obviously promote the proliferation of the granulation tissues and fibroblasts. The number of AgNORs granules in fibrablasts were 3.73 ±0.52 in the buy group and 2.11 ±0.31 in control group (P < 0.05). CONCLUSION: bFGF could promote the growth of granulation tissues and had a strong promotion on the wound healing in palatal perforation. It was helpful in repairing palatal perforation.     

Effects of bilberry anthocyanins on MMP2 and collagen I in human fetal scleral fibroblast in vitro

AIM:To investigate the effects of bilberry anthocyanins on matrix metalloproteinase 2 (MMP2) and collagen type I (collagen I) expression in human fetal scleral fibroblasts (HFSF) in vitro and to provide experimental data for the prevention and treatment of myopia by bilberry anthocyanins. METHODS:HFSF were treated with bilberry anthocyanins at different concentrations (0, 10^-6, 10^-5, 10^-4, 10^-3, 10^-2, 10^-1 and 1 g/L). The effects of bilberry anthocyanins on the viability of HFSF for different time (6 h, 8 h, 12 h, 24 h and 48 h) were measured by MTT assay. The cell cycle distribution and apoptosis of HFSF with highest viability (10^-1 g/L bilberry anthocyanins for 12 h) were analyzed by flow cytometry. The mRNA expression of MMP2, COL1A1 and COL1A2 in the HFSF was detected by RT-qPCR. The protein expression of MMP2 and collagen I was determined by Western blot. RESULTS:The cell viability was the highest after treatment with bilberry anthocyanins at a concentration of 10^-1 g/L for 12 h. Compared with blank control group, 10^-1 g/L bilberry anthocyanin group showed increased cell numbers in S and G₂ phases (P<0.05), but no significant difference of the apoptotic rate was observed. The mRNA expression of MMP2 was decreased (P<0.05), and that of COL1A1 was increased (P<0.05). No significant difference of COL1A2 expression was seen. The protein expression of MMP2 was decreased (P<0.05), and collagen I protein was increased (P<0.05). CONCLUSION:Bilberry anthocyanins inhibit the expression of MMP2 and increase the expression of collagen I in the HFSF.     

Protective effects of riboflavin on diabetic nephropathy in STZ-induced diabetic rats

AIM: To explore the protective effects of riboflavin on the kidney in streptozotocin (STZ)-induced diabetic rats. METHODS: Male Sprague-Dawley rats were randomly divided into 3 groups: normal control group, diabetic model group and riboflavin-treated group. Diabetes was induced by a single injection of STZ (dissolved in 0.01 mol/L citrate buffer, pH 4.5, 65 mg/kg, ip) in rats. The biochemical methods were used to measure the contents of urine protein and malondialdehyde in the kidney, and the activities of superoxide dismutase (SOD) and catalase (CAT) in serum and renal tissues. Furthermore, the protein expression of TGF-β1 and plasminogen activator inhibitor-1(PAI-1) in renal cortex was detected by Western blotting. The morphological changes of renal tissue were observed under microscope.RESULTS: Compared to the diabetic model group, riboflavin significantly increased the activities of SOD and CAT (P<0.01) in the serum and renal tissues, and decreased the contents of urine protein and MDA (P<0.01) in the renal tissues in riboflavin-treated group. The levels of TGF-β1 and PAI-1 in the renal cortex were dramatically decreased in the treated diabetic rats compared to the diabetic model rats (P<0.01).CONCLUSION: Riboflavin inhibits the protein expression of TGF-β1 and PAI-1 in renal tissue of STZ-induced diabetic rats. Riboflavin may alleviate the pathologic changes and play an important protective role in diabetic kidneys.     

Activation of α7nAChR promotes wound healing in diabetic mice by suppressing TNF-α expression

AIM: To explore the role of α7 nicotinic acetylcholine receptor (α7nAChR)-specific agonist PNU-282987 in promoting wound healing in diabetic mice by suppressing the expression of tumor necrosis factor α （TNF-α）.METHODS: A model of incised wound was established in diabetic mice or normoglycaemic mice (control). Skin samples were taken on 1 d, 3 d, 5 d, 10 d, 14 d and 21 d post-injury (5 mice in each posttraumatic interval). The numbers of macrophages and fibroblasts, the expression of TNF-α and the deposition of collagen were detected by the methods of immunohistochemistry, Western blotting and Masson staining, respectively. After incised wound was performed in the diabetic mice, PNU-282987 was applied by intraperitoneal injection at suitable posttraumatic interval. The above indexes were investigated again.RESULTS: Compared with control group, the diabetic mice presented delayed wound healing. In diabe-tic mice, the infiltration of macrophages and the expression of TNF-α were significantly reduced in the early phase during wound healing, while they were significantly increased from 5 d post-injury. Besides, from 5 d to 21 d post-injury, the wounds in diabetic mice showed decreased number of fibroblasts and deposition of collagen. From 5 d post-injury, PNU-282987 was applied to diabetic mice, which significantly down-regulated the expression of TNF-α, and increased the number of fibroblasts and the content of collagen in the wounds, eventually promoted wound healing.CONCLUSION: Inflammatory reactions delay wound healing in diabetic mice. Activation of α7nAChR promotes wound healing in diabetic mice by suppressing the expression of TNF-α.     

Inhibitory effect of arsenic trioxide on the proliferation of airway fibroblast and the expression of c-myc and c-sis in vitro

AIM: To study the effects of arsenic trioxide (As2O3) on mouse airway fibroblast (FB) proliferation and expression of c-myc and c-sis in cultured FB. METHODS: The cultured FB was divided into seven groups. NS, dexamethasone (1.0 μmol/L) and As2O3 at concentrations of 0.25, 0.5, 1.0, 2.0, 4.0 μmol/L were instilled respectively into cultured FB. The effects of different concentration of As2O3 and dexamethasone on cell growth were observed at 1, 3, 5 and 7 day. The changes of cell cycle and the positive expression rate of c-myc and c-sis were examined by flow cytometry (FCM). RESULTS: Various concentrations of As2O3 significantly inhibited the proliferation of FB in vitro and showed a dose-dependent and time-dependent tendency. Data from FCM indicated that G1-phase cell percentage increased and G2/M-phase cell percentage decreased with the increase in As2O3 concentrations. The expression of c-myc and c-sis was significantly inhibited by As2O3 (2.0, 4.0 μmol/L). CONCLUSION: As2O3 inhibits FB proliferation by downregulating the expression of c-myc and c-sis.     

Effects of probucol on the proliferation of rat vascular smooth muscle cells stimulated by basic fibroblast growth factor and hydrogen peroxide

AIM: To investigate the effect of probucol on proliferation of rat vascular smooth muscle cells(VSMC) stimulated by basic fibroblast growth factor (bFGF) and/or hydrogen peroxide(H₂O₂). METHODS: Effects of probucol on VSMC proliferation and DNA synthesis stimulated by bFGF and/or H₂O₂ were observed by means of MTT test, cell number count and [3H]-TdR incorporation. RESULTS: ①Probucol significantly inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, with dosage-dependent manner. Cell number, A value and [3H]-TdR incorporation in group probucol+bFGF and group probucol+H₂O₂ were reduced by 40.0%, 39.1%, 45.5% and 46.9%, 45.0%, 39.5%, respectively, compared with group bFGF and group H₂O₂ (P＜0.05, P＜0.01, respectively). ②Pretreatment of VSMC with probucol for 24 h prior to bFGF and/or H₂O₂ stimulation exhibited significant inhibiton of VSMC proliferation and DNA synthesis, but after prestimulation by bFGF and/or H₂O₂ for 24 h, probucol had no influence on VSMC proliferation and DNA synthesis (P＞0.05). CONCLUSION: Probucol dramatically inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, but had no inhibitory effect on the cell proliferation prestimulated by bFGF and /or H₂O₂.