Effects of haem oxygenase-1 expression on oxidative injury and biological behaviours of rat dermal fibroblasts.

OBJECTIVE: This study investigated the effects of high haem oxygenase-1 (HO-1) expression on oxidative injury and the biological behaviours of rat dermal fibroblasts, under high glucose conditions. METHOD: Rat dermal fibroblasts were cultured in normal glucose (1.0g/l), high glucose (4.5g/l) or haemin (5µm). A bilirubin kit, real-time polymerase chain reaction (RT-PCR) and Western blotting measured the protease activity, mRNA, and protein levels of HO-1, respectively. An enzyme-linked immunosorbent assay (ELISA) kit measured media levels of 8-hydroxydeoxyguanosine (8-OHdG), reactive oxygen species (ROS) and collagen (hydroxyproline) secretion. Cell proliferation was measured using flow cytometry. Cell apoptosis was measured using Hoechst 33258 staining and flow cytometry. The transwell method and scratch test evaluated cell migration. RESULTS: HO-1 expression exhibited a time-dependent change that was lowest in the high glucose (HG) group at 96 hours compared with the normal glucose (NG) group. In the HG group, the 8-OHdG, ROS and cell apoptosis were increased, and collagen secretion, cell proliferation and cell migration (horizontal and vertical) were decreased compared with the NG group at 96 hours. Haemin treatment sustained high HO-1 expression for at least 96 hours, and the cells exhibited decreased 8-OHdG and ROS, increased collagen synthesis, improved proliferation and migration ability, and decreased apoptosis in the NG and haemin (NH) group/HG and haemin (HH) group compared with the NG/HG groups. These cells recovered from oxidative injury and biological behaviours dysfunction. CONCLUSION: Haemin induces HO-1 expression in fibroblasts and it may influence the oxidative injury and biological behaviours of fibroblasts. These findings suggest that HO-1 may accelerate the healing of diabetic wounds via alleviation of oxidative injury and improvement of biological behaviours of fibroblasts.

[Diagnostic criteria for late-onset hypogonadism: A preliminary study].

OBJECTIVE: To determine the stability of androgen indexes by analyzing the relationship of androgen indexes with the results of late-onset hypogonadism (LOH) questionnaire investigations, and offer some reference for the application of the diagnostic criteria for LOH released by The Chinese Society of Andrology in 2009. METHODS: This study included 1 003 males aged 40 years or older who had accomplished the questionnaires of Androgen Deficiency in Aging Males (ADAM), Aging Males' Symptoms Scale (AMS), and International Index of Erectile Function-5 (IIEF-5). We evaluated the correlation of androgen indexes with the results of the questionnaire investigation, repeated the examination of androgen indexes for the subjects with total testosterone (TT) ≤11.5 nmol/L after an average of 1.5 years, and analyzed the factors inducing changes of androgen indexes. RESULTS: Free testosterone index (FTI) ≤ 0.42 (OR, 1.369) and calculated free testosterone (cFT) ≤ 0.3 nmol/L (OR, 1.302) were considered as the risk factors of LOH in AMS, and so were testosterone secretion index (TSI) ≤ 2.8 nmol/IU (OR, 1.679) and cFT ≤ 0.3 nmol/L (OR, 1.371) in IIEF-5. Paired t-test on the results of the examination performed twice showed significant differences in the levels of TT, TSI, cFT, and FT (P<0.05). CONCLUSIONS: Decreased testosterone may cause the diversity of LOH symptoms and hence the fluctuation of androgens. Therefore, the diagnosis of LOH depends on androgen indexes, varied symptoms in the questionnaires, and relief of the symptoms after testosterone therapy.

[Association of serum testosterone with atherosclerosis in middle-aged and elderly men].

OBJECTIVE: To investigate the association between the level of serum testosterone and atherosclerosis in middle-aged and elderly men. METHODS: We conducted a population-based study of 413 males aged 40-75 years in a community in Guangzhou. We obtained the sociodemographic characteristics, clinical data, physical measurements, and laboratory results of sex hormones, blood glucose, and blood lipid of the subjects. We also measured the carotid intima media thickness (CIMT) by color Doppler ultrasonography. RESULTS: The subjects were divided into a carotid atherosclerosis (CAS) group (CIMT ≥ 0.9 mm) and a non-CAS group (CIMT < 0.9 mm). The medians of free testosterone (FT) were 57.41 and 59.72 pmol/L in the CAS and non-CAS groups, respectively (P = 0.005), and no significant difference was found between the two groups in total testosterone (TT). The levels of serum FT and TT were negatively correlated to CIMT, with Spearman's rank correlation coefficients of -0.126 (P = 0.011) and -0.188 (P < 0.001), respectively. The incidence rates of CAS were 23.30, 13.46, 17.48, and 7.77% in the Q1, Q2, Q3, and Q4 groups, respectively according to the quartile of FT (P for trend = 0.008) and 17.48, 18.27, 16.50, and 9.71% respectively according to the quartile of TT (P for trend = 0.116). Based on the quartile of FT and after adjustment for age, waist circumference, systolic blood pressure, and HbAlc, the risk of CAS was significantly increased in the Q1 group as compared with Q4 (OR = 2.491, 95% CI 1.01-6.149), but no statistically significant differences were observed according to the quartile of TT. CONCLUSION: A low serum FT level may be a risk factor of atherosclerosis in Chinese men aged 40 years or older.

[Effects of matrix metallopeptidase 9 on the proliferation, apoptosis, type I and III procollagen synthesis of rat dermal fibroblasts].

OBJECTIVE: To explore the effects of matrix metallopeptidase 9 (MMP-9) on the proliferation, apoptosis, type I and III procollagen synthesis of rat dermal fibroblasts. METHODS: Lipopolysaccharide (LPS) at 0.1 and 1.0 µg/ml was used to stimulate fibroblasts to up-regulate the expression of MMP-9 for 18 and 48 h. And then RNA interference was employed to inhibit the high expression of MMP-9. Cell proliferation was tested by CCK-8, cell apoptosis by flow cytometry and type I and III procollagen expressions by quantitative reverse transcriptase PCR (qRT-PCR). RESULTS: The MMP-9 expression of fibroblasts increased after the stimulation of LPS. And the 1.0 µg/ml LPS stimulation of 48 hours was 14.25 times of mRNA expression and 2.31 times of protein expression versus that of the normal group (both P < 0.05). The RNA interference obviously inhibited the high expression of MMP-9. The mRNA expression was 1/8 and protein expression 1/3 (both P < 0.05) as compared with the control group. Cell proliferation decreased with the rising expression of MMP-9 to some extent [(1.08 ± 0.08) vs (1.18 ± 0.09), P < 0.05] and improved after the inhibition of high expression of MMP-9 [(1.78 ± 0.17) vs (1.53 ± 0.15), P < 0.01]. There was no change of apoptosis accompanied with high expression of MMP-9 (P > 0.05). Apoptosis decreased after the inhibition of high expression of MMP-9 for 48 hours (3.53% ± 0.22% vs 4.47% ± 0.46%, P < 0.05). The synthesis of type I procollagen was the same no matter up-regulation or down-regulation of MMP-9 expression (P > 0.05). As the expression of MMP-9 increased, the synthesis of type III procollagen decreased (P < 0.01), but not increased by 1.02 folds after the inhibited expression of MMP-9 (P > 0.05). CONCLUSION: MMP-9 can affect the biological behaviors of rat dermal fibroblasts.

Potential Novel Serum Metabolic Markers Associated With Progression of Prediabetes to Overt Diabetes in a Chinese Population.

Background: Identifying the metabolite profile of individuals with prediabetes who turned to type 2 diabetes (T2D) may give novel insights into early T2D interception. The purpose of this study was to identify metabolic markers that predict the development of T2D from prediabetes in a Chinese population. Methods: We used an untargeted metabolomics approach to investigate the associations between serum metabolites and risk of prediabetes who turned to overt T2D (n=153, mean follow up 5 years) in a Chinese population (REACTION study). Results were compared with matched controls who had prediabetes at baseline [age: 56 ± 7 years old, body mass index (BMI): 24.2 ± 2.8 kg/m2] and at a 5-year follow-up [age: 61 ± 7 years old, BMI: 24.5 ± 3.1 kg/m2]. Confounding factors were adjusted and the associations between metabolites and diabetes risk were evaluated with multivariate logistic regression analysis. A 10-fold cross-validation random forest classification (RFC) model was used to select the optimal metabolites panels for predicting the development of diabetes, and to internally validate the discriminatory capability of the selected metabolites beyond conventional clinical risk factors. Findings: Metabolic alterations, including those associated with amino acid and lipid metabolism, were associated with an increased risk of prediabetes progressing to diabetes. The most important metabolites were inosine [odds ratio (OR) = 19.00; 95% confidence interval (CI): 4.23-85.37] and carvacrol (OR = 17.63; 95% CI: 4.98-62.34). Thirteen metabolites were found to improve T2D risk prediction beyond eight conventional T2D risk factors [area under the curve (AUC) was 0.98 for risk factors + metabolites vs 0.72 for risk factors, P < 0.05]. Interpretations: Use of the metabolites identified in this study may help determine patients with prediabetes who are at highest risk of progressing to diabetes.

A greater glycemic load reduction was associated with a lower diabetes risk in pre-diabetic patients who consume a high glycemic load diet.

Few prospective studies evaluating the association between dietary glycemic load (GL) and diabetes have accounted for changes in GL. However, the diet of patients could be modified in response to an awareness of pre-diabetes. The aim of this study was to examine the longitudinal associations between changes in GL and the incidence of diabetes among pre-diabetic patients. We hypothesized that subjects with low and high baseline GL would show different correlations with diabetes. A total of 493 pre-diabetic patients (142 men and 351 women) between 40 and 79 years of age were included in this study. Dietary records and oral glucose tolerance tests were conducted every year. The participants were divided into low- and high-GL groups based on baseline GL. During a median 4 years of follow-up, 108 incident cases of diabetes were identified. Among participants with a high baseline GL, the incidence of diabetes increased with decreasing GL reduction, and the multivariate-adjusted HR (95% CI) was 2.34 (1.27-4.29) when comparing the lowest to the highest tertiles; however, among those with a low baseline GL, no significant association was observed. Regardless of baseline GL status, the incidence of diabetes was higher in individuals with a high follow-up GL than in those with a low follow-up GL, and the multivariate-adjusted HR (95% CI) was 1.64 (1.09-2.45). In conclusion, a greater GL reduction was associated with a lower diabetes risk in pre-diabetic patients with a high dietary GL. In patients with pre-diabetes and a low dietary GL, further reductions in GL did not show any additional effects.

Glycemic load is associated with diabetes and prediabetes among middle-aged and elderly adults in Guangzhou, China.

BACKGROUND AND OBJECTIVES: Previous studies have obtained conflicting findings regarding the possible associations between glycemic load (GL) indices and diabetes. In the present study, we examined cross-sectional associations between several GL indices, including the total dietary GL, the energy-adjusted GL, and the prevalence of abnormal glucose metabolism, including prediabetes and diabetes. METHODS AND STUDY DESIGN: This study was conducted in Guangzhou, China from July 2011 to December 2011. It included 2,022 participants (602 men and 1,420 women), between 45 and 75 years of age. The prevalence of abnormal glucose metabolism was compared across the quartiles of GL indices to discover any potential linear correlations. Stratified analysis was conducted according to the body mass index (BMI) and waist circumference (WC) measurements. RESULTS: Energy-adjusted GL was positively associated with the prevalence of diabetes and the multivariable-adjusted estimate of the OR comparing the highest versus the lowest quartile was 2.50 (95% CI, 1.49-4.19). For the stratified analysis by sex, BMI or WC, similar associations were observed. For the overweight and obese (BMI ≥24.0 kg/m2) or centrally obese (WC ≥85 cm for men or ≥80 cm for women) participants, compared to participants in the lowest quartile of energy-adjusted GL, those in the highest quartile showed an increased risk of abnormal glucose metabolism. The OR estimates were 2.25 (95% CI: 1.45-3.52) and 1.54 (95% CI: 1.06-2.25), respectively. CONCLUSIONS: High dietary energy-adjusted GL is associated with the prevalence of diabetes as well as abnormal glucose metabolism among middle-aged and elderly adults.

Association between obesity measures and albuminuria: A population-based study.

AIMS: The effects of obesity on the micro vascular diseases have drawn much attention. The aim of the study was to investigate the relationship between obesity measures and albuminuria in Chinese population. METHODS: We conducted a population-based cross-sectional study in 8600 subjects aged 40 years or older from a community in Guangzhou. Urinary albumin excretion and creatinine were measured and urinary albumin-to-creatinine ratio (ACR) was calculated as urinary albumin divided by creatinine. Low-grade albuminuria was classified as the highest quartile of ACR in participants without increased urinary albumin excretion. Increased urinary albumin excretion was defined according to the ACR ranges greater or equal than 30 mg/g. RESULTS: Pearson's correlation analysis and multivariate linear regression analysis revealed that body mass index (BMI), waist circumference and body fat content were significantly correlated with ACR (all P<0.01). Prevalence of low-grade albuminuria and increased urinary albumin excretion gradually increased across the BMI, waist circumference and body fat content quartiles (all P for trend<0.0001). Compared with participants in quartile 1 of BMI, waist circumference and body fat content, participants in quartile 4 had increased prevalence of low-grade albuminuria and increased urinary albumin excretion in logistic regression analysis after adjustment for age, sex, physical activity, fasting plasma glucose, triglycerides, low-density lipoprotein cholesterol and HbA1c (all P<0.05). CONCLUSION: Obesity measures are associated with urinary albumin excretion in middle-aged and elderly Chinese.

Changes in biological behaviors of rat dermal fibroblasts induced by high expression of MMP9.

BACKGROUND: The high level of matrix metalloproteinase 9 (MMP9) is thought to slow down the healing of diabetic foot ulcers. Whether it can influence the biological behaviors of skin fibroblasts and affect wound healing is still unclear. The present study aimed to observe changes in the biological behaviors of rat dermal fibroblasts induced by high expression of MMP9 and to clarify the possible mechanisms of wound healing for diabetic foot. METHODS: A cell model of skin fibroblast with high expression of MMP9 was established by co-culture of high glucose (22.0 mmol/L) and homocysteine (100 µmol/L). A control group was incubated with normal glucose (5.5 mmol/L). Realtime PCR, ELISA and gelatin zymography were used to detect the MMP9 mRNA, protein expression and activity of MMP9. Flow cytometry, CCK-8, ELISA assay, scratch test and transwell were used to detect cell proliferation, viability, collagen (hydroxyproline) secretion, horizontal migration and vertical migration of cells. The data were expressed as mean±SD. P value less than 0.05 was considered statistically significant. RESULTS: The expression of MMP9 mRNA, protein levels and the activity of MMP9 were much higher in the high MMP9 group than in the control group (7.05±1.02 vs. 1.00±0.00, 206.9±33.6 pg/mL vs. 40.4±5.9 pg/mL, and 1.47±0.13 vs. 0.57±0.12, respectively, P<0.01). The proportion of S-phase cells, proliferation index, cell viability, collagen (hydroxyproline) secretion, horizontal migration rate and the number of vertical migration cells were lower in the high MMP9 group than in the control group (P<0.01). CONCLUSION: Fibroblasts with a high expression of MMP9 decreased proliferation, activity, secretion and migration of collagens, suggesting that MMP9 may inhibit the biological behaviors of fibroblasts.

Cationic star-shaped polymer as an siRNA carrier for reducing MMP-9 expression in skin fibroblast cells and promoting wound healing in diabetic rats.

BACKGROUND: Excessive expression of matrix metalloproteinase-9 (MMP-9) is deleterious to the cutaneous wound-healing process in the context of diabetes. The aim of the present study was to explore whether a cationic star-shaped polymer consisting of ß-cyclodextrin (ß-CD) core and poly(amidoamine) dendron arms (ß-CD-[D3]7) could be used as the gene carrier of small interfering RNA (siRNA) to reduce MMP-9 expression for enhanced diabetic wound healing. METHODS: The cytotoxicity of ß-CD-(D3)7 was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MMT) method in the rat CRL1213 skin fibroblast cell line. The transfection efficiency of ß-CD-(D3)7/MMP-9-small interfering RNA (siRNA) complexes was determined by confocal microscopy and flow cytometry. Quantitative real time (RT) polymerase chain reaction was performed to measure the gene expression of MMP-9 after the transfection by ß-CD-(D3)7/MMP-9-siRNA complexes. The ß-CD-(D3)7/MMP-9-siRNA complexes were injected on the wounds of streptozocin-induced diabetic rats. Wound closure was measured on days 4 and 7 post-wounding. RESULTS: ß-CD-(D3)7 exhibited low cytotoxicity in fibroblast cells, and easily formed the complexes with MMP-9-siRNA. The ß-CD-(D3)7/MMP-9-siRNA complexes were readily taken up by fibroblast cells, resulting in the downregulation of MMP-9 gene expression (P<0.01). Animal experiments revealed that the treatment by ß-CD-(D3)7/MMP-9-siRNA complexes enhanced wound closure in diabetic rats on day 7 post-wounding (P<0.05). CONCLUSION: ß-CD-(D3)7 may be used as an efficient carrier for the delivery of MMP-9-siRNA to reduce MMP-9 expression in skin fibroblast cells and promote wound healing in diabetic rats.

Effect of intensive nursing education on the prevention of diabetic foot ulceration among patients with high-risk diabetic foot: a follow-up analysis.

The aim of the study was to discuss the effect of intensive nursing education on the prevention of diabetic foot ulceration among patients at high risk for diabetic foot. One hundred eighty-five diabetes patients at high risk for foot diseases were enrolled in this study and provided with intensive nursing education, including individualized education about diabetes mellitus and diabetic foot diseases, instruction in podiatric care (the right way of washing the foot, the care of foot skin, appropriate choice of shoes and socks, intense examinations and records of feet by patients themselves every day, and the assistant management of calluses). Study subjects were followed up for 2 years. Once the foot ulceration developed, the inducing factors of foot ulceration were inquired about, the ulcers were evaluated, and the incidence of foot ulceration was analyzed before and after the intensive nursing education according to self-paired data. Results showed there were highly statistically significant improvements in the intensive treatment group compared with the control group in plasma glucose, blood pressure, and high-density lipoprotein cholesterol levels. More important is that intensive nursing education helps to prevent diabetic foot ulceration and to decrease the rate of amputation among patients at high risk for diabetic foot.

The biological behaviors of rat dermal fibroblasts can be inhibited by high levels of MMP9.

AIMS: To explore the effects of the high expression of MMP9 on biological behaviors of fibroblasts. METHODS: High glucose and hyperhomocysteine were used to induce MMP9 expression in skin fibroblasts. Cell proliferation was detected by flow cytometry and cell viability by CCK-8. ELISA assay was used to detect collagen (hydroxyproline) secretion. Scratch test was employed to evaluate horizontal migration of cells and transwell method to evaluate vertical migration of cells. RESULTS: The mRNA and protein expressions of MMP9 and its protease activity were significantly higher in cells treated with high glucose and hyperhomocysteine than those in control group. At the same time, the S-phase cell ratio, proliferation index, cell viability, collagen (hydroxyproline) secretion, horizontal migration rate, and the number of vertical migration cells decreased in high-glucose and hyperhomocysteine-treated group. Tissue inhibitor of metalloproteinase 1 (TIMP1), which inhibits the activity of MMP9, recovered the above biological behaviors. CONCLUSIONS: High expression of MMP9 in skin fibroblasts could be induced by cultureing in high glucose and hyperhomocysteine medium, which inhibited cell biological behaviors. Inhibitions could be reversed by TIMP1. The findings suggested that MMP9 deters the healing of diabetic foot ulcers by inhibiting the biological behaviors of fibroblasts.

Activation of skin renin-angiotensin system in diabetic rats.

The renin-angiotensin system (RAS) is reportedly involved in chronic diabetic complications such as diabetic nephropathy, but changes of the RAS in diabetic skin remain unknown. The aim of this study was to investigate the expression of angiotensin (Ang) II and its type 1 (AT1) and type 2 (AT2) receptors in diabetic skin tissues, and explore the relationship between the local RAS and pathological changes of diabetic skin. Our results showed that thinning of epidermis, degeneration of collagen, fracture of dermal layer, and atrophy/disappearance of subcutaneous fat were observed in diabetic skin. The expression level of AngII was increased in diabetic skin tissues compared to that in controls. mRNA and protein expression of AT1 receptor were also increased while the level of AT2 receptor decreased; the relative expression of AT1 to AT2 receptors was approximately threefold higher in diabetes than in controls. Furthermore, in the culture medium of primary cultured fibroblasts from diabetic skin, the concentration of AngII was significantly higher than that of normal control. The mRNA and protein expression of AT1 receptor was also increased in fibroblasts of diabetic skin compared to controls, while the protein expression of AT2 receptor was decreased. Taken together, our results suggest that the local RAS system is activated in diabetic skin and AngII receptor is likely to mediate the pathological changes of diabetic skin.