Reproductive toxicity of perchlorate in rats.

Perchlorate, as an oxidizer, has many applications such as explosives and pyrotechnics, especially in rocket propellants and missile motors. Because it was found in water including wells and drinking water in the US, its effect on human health was being noted. However, the reproductive toxic effect on perchlorate is still unclear. In present study, the effects of repeated exposure to perchlorate on reproductive toxicity were evaluated in Wistar rats. The rats were treated orally with perchlorate at doses of 0.05, 1.00 or 10.00 mg/kg body weight (b.w.) daily for 8 weeks. The levels of T3 and T4 hormones in the rat serum were detected by radioimmunoassay kit. The indexes of reproduction, percentage of organ in body weight (%) and frequency of abnormal sperm cells were also analyzed in this study. DNA damage in testicular cells was evaluated by Comet assay. The levels of MDA, GSH and SOD were examined in testicle tissues of rats by ELISA. The expression of c-fos and fas protein was examined in testicle tissues by immunohistochemistry. The results showed that perchlorate did not affect the body weight of rats. Perchlorate also significantly decreased indexes of live birth and weaning in the groups of 1.00 and 10.00 mg/kg, and viability index only in the 10.00 mg/kg group (P < 0.05). Perchlorate also significantly decreased the serum level of T3 in male rats of 1.00 and 10.00 mg/kg groups, increased the rate of sperm abnormality (10.00 mg/kg), potentially caused DNA damage in testicular cells and altered the status of oxidative stress in male rats. In addition, because of the increase in the expression of fas and c-fos protein in testicle tissues, perchlorate could induce apoptosis in spermatogenesis. Thus, these findings indicate that perchlorate could cause DNA damage in testicular tissues and reduce testicular spermatogenic ability, resulting in reproductive toxicity.

MicroRNA-138 Aggravates Inflammatory Responses of Macrophages by Targeting SIRT1 and Regulating the NF-κB and AKT Pathways.

BACKGROUND/AIMS: With increased understanding of sepsis, mortality is decreasing. However, there is still a lack of effective therapeutic strategy. The inflammatory response of macrophages is critical during sepsis. METHODS: Macrophages were stimulated with LPS. Western blotting and qRT-PCR were used to detect inflammatory responses. Then, the inhibitor of microRNA-138 was transfected and Western blotting, qRT-PCR, H&E staining and ELISA were used to verify the role of microRNA-138 in inflammation. Then target gene prediction databases were used to predict the potential target of microRNA-138. Both animal and cell models under LPS challenges were established to verify the regulation of SIRT1 and microRNA-138 during inflammation. RESULTS: The present study showed that microRNA-138 was increased in macrophages stimulated with LPS. Additionally, the NF-κB and AKT pathways were both activated. The pre-treatment of microRNA-138 inhibitor decreased inflammatory factors, downregulated the NF-κB pathway, activated the AKT pathway and protected against organ damage in mice challenged with LPS. SIRT1 was demonstrated as a potential target of microRNA-138In macrophages stimulated with LPS, the inhibition effect of microRNA-138 inhibitor on inflammation was lost by SIRT1 siRNA pre-treatment. In the animal model, the protective effect of microRNA-138 antagomir disappeared in SIRT1 knockout mice. CONCLUSION: We demonstrated that miR-138 participated in the inflammatory process by inhibiting SIRT1 and activating the NF-κB pathway.

Melatonin prevents acute kidney injury in severely burned rats via the activation of SIRT1.

Acute kidney injury (AKI) is a common complication after severe burns. Melatonin has been reported to protect against multiple organ injuries by increasing the expression of SIRT1, a silent information regulator that regulates stress responses, inflammation, cellular senescence and apoptosis. This study aimed to investigate the protective effects of melatonin on renal tissues of burned rats and the role of SIRT1 involving the effects. Rat severely burned model was established, with or without the administration of melatonin and SIRT1 inhibitor. The renal function and histological manifestations were determined to evaluate the severity of kidney injury. The levels of acetylated-p53 (Ac-p53), acetylated-p65 (Ac-p65), NF-κB, acetylated-forkhead box O1 (Ac-FoxO1), Bcl-2 and Bax were analyzed to study the underlying mechanisms. Our results suggested that severe burns could induce acute kidney injury, which could be partially reversed by melatonin. Melatonin attenuated oxidative stress, inflammation and apoptosis accompanied by the increased expression of SIRT1. The protective effects of melatonin were abrogated by the inhibition of SIRT1. In conclusion, we demonstrate that melatonin improves severe burn-induced AKI via the activation of SIRT1 signaling.

Identification of sirtuin 1 as a promising therapeutic target for hypertrophic scars.

BACKGROUND AND PURPOSE: Sirtuin1 (SIRT1), the founding member of mammalian class III histone deacetylases, is reported to be a drug target involved in fibrotic diseases. However, whether it is an effective drug target in hypertrophic scar treatment is still not known. EXPERIMENTAL APPROACH: In the present study, we observed that SIRT1 localized to both the epidermis and the dermis of skin tissues by immunohistochemistry. After knock-down of SIRT1 by shRNA or up-regulating SIRT1 by resveratrol, the expression of α-SMA, Col1 and Col3 in fibroblasts were detected by western blots. A mouse excision wound healing model was used to observe the changes in collagen fibre associated with the different expression levels of SIRT1. KEY RESULTS: SIRT1 expression was inhibited in hypertrophic scar tissue. The down-regulation of SIRT1 resulted in an increased expression of α-SMA, Col1 and Col3 in hypertrophic scar-derived fibroblasts. In contrast, the up-regulation of SIRT1 not only inhibited the expression of α-SMA, Col1 and Col3 in hypertrophic scar-derived fibroblasts but also blocked the activation of TGFß1-induced normal skin-derived fibroblasts. In the mouse model of wound healing, the deletion of SIRT1 resulted in denser collagen fibres and a more disordered structure, whereas resveratrol treatment led to a more organized and thinner collagen fibre, which was similar to that observed during normal wound healing. CONCLUSIONS AND IMPLICATIONS: The results revealed that SIRT1 negatively regulates TGFß1-induced fibroblast activation and inhibits excessive scar formation and is, therefore, a promising drug target for hypertrophic scar formation.

Acute downregulation of miR-155 at wound sites leads to a reduced fibrosis through attenuating inflammatory response.

Fibrosis, tightly associated with wound healing, is a significant symptomatic clinical problem. Inflammatory response was reported to be one of the reasons. MiR-155 is relatively related with the development and requirement of inflammatory cells, so we thought reduce the expression of miR-155 in wound sites could improve the quality of healing through reduce inflammatory response. To test this hypothesis, locally antagonizing miR-155 by directly injecting antagomir to wound edge was used to reduce the expression of miR-155. We found wounds treated with miR-155 antagomir had an obvious defect in immune cells requirements, pro-inflammatory factors IL-1ß and TNF-α reduced while anti-inflammatory factor IL-10 increased. With treatment of miR-155 antagomir, the expression of α-smooth muscle actin (α-SMA), Col1 and Col3 at wound sites all reduced both from mRNA levels and protein expressions. Wounds injected with antagomir resulted in the structure improvement of collagen, the collagen fibers were more regularly arranged. Meanwhile the rate of healing did not change significantly. These results provide direct evidences that miR-155 play an important role in the pathogenesis of fibrosis and show that miR-155 antagomir has the potential therapy in prevention and reduction of skin fibrosis.

MicroRNA-21 regulates hTERT via PTEN in hypertrophic scar fibroblasts.

BACKGROUND: As an important oncogenic miRNA, microRNA-21 (miR-21) is associated with various malignant diseases. However, the precise biological function of miR-21 and its molecular mechanism in hypertrophic scar fibroblast cells has not been fully elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative Real-Time PCR (qRT-PCR) analysis revealed significant upregulation of miR-21 in hypertrophic scar fibroblast cells compared with that in normal skin fibroblast cells. The effects of miR-21 were then assessed in MTT and apoptosis assays through in vitro transfection with a miR-21 mimic or inhibitor. Next, PTEN (phosphatase and tensin homologue deleted on chromosome ten) was identified as a target gene of miR-21 in hypertrophic scar fibroblast cells. Furthermore, Western-blot and qRT-PCR analyses revealed that miR-21 increased the expression of human telomerase reverse transcriptase (hTERT) via the PTEN/PI3K/AKT pathway. Introduction of PTEN cDNA led to a remarkable depletion of hTERT and PI3K/AKT at the protein level as well as inhibition of miR-21-induced proliferation. In addition, Western-blot and qRT-PCR analyses confirmed that hTERT was the downstream target of PTEN. Finally, miR-21 and PTEN RNA expression levels in hypertrophic scar tissue samples were examined. Immunohistochemistry assays revealed an inverse correlation between PTEN and hTERT levels in high miR-21 RNA expressing-hypertrophic scar tissues. CONCLUSIONS/SIGNIFICANCE: These data indicate that miR-21 regulates hTERT expression via the PTEN/PI3K/AKT signaling pathway by directly targeting PTEN, therefore controlling hypertrophic scar fibroblast cell growth. MiR-21 may be a potential novel molecular target for the treatment of hypertrophic scarring.

[A review of research on Schmallenberg virus].

Schmallenberg virus (SBV), a novel orthobunyavirus, was first isolated in 2011. SBV preferentially infects the central nervous system of cattle and sheep and causes fever, diarrhea, a drop in milk yields, congenital malformations and stillbirths. Until June 2014, more than 200 scientific publications regarding SBV have been published. Although more than 20 articles on SVB were published in China, most of these articles provided only a brief introduction of the disease without fully discussing the associated disease characteristics. As a new disease, it has been made a focus of the National Research Center for Exotic Animal Diseases at the China Animal Health and Epidemiology Center. In this review, in order to provide a reference for research into SBV in China, we have reviewed the state of current research progress on the etiology, diagnosis and epidemiology of SBV, and vaccine development.

[Impact of resting heart rate on new-onset diabetes in population without hypertension].

OBJECTIVE: To investigate the impact of resting heart rate (RHR) on new-onset diabetes (NOD) in population without hypertension. METHODS: This prospective cohort study was performed in 2006 and 2007 and screened 101 510 participants. All subjects were employees of the Kailuan Group, a state-run coal mining company. The observation cohort included 48 926 subjects with normal fasting blood glucose (FBG) <7.0 mmol/L, no history of diabetes, complete FBG and RHR examination data, systolic blood pressure <140 mm Hg (1 mm Hg = 0.133 kPa) , diastolic blood pressure <90 mm Hg, no history of hypertension, and no use of hypoglycemic agents or antihypertensive drugs.We excluded participants without a health examination in 2008-2009 or 2010-2011 and those with incomplete examination data. A total of 29 910 participants were included in the final analysis. The observation population was divided into four groups according to RHR data collected during 2006-2007 health examinations: quartile 1 (RHR<63 beats/min) ; quartile 2 (63 beats/min ≤ RHR<70 beats/min) ; quartile 3 (70 beats/min ≤ RHR<75 beats/min) ; quartile 4 ( RHR ≥ 75 beats/min). Kaplan-Meier analysis was used to calculate the incidence of NOD. The relationship between RHR and NOD was estimated using Cox proportional hazard analysis. RESULTS: The incidences of NOD/1000 person-years for the above quartiles of RHR were 11.22, 13.58, 13.96, and 17.55, respectively in the total observational population; the corresponding incidences were 12.17, 15.20, 16.08, 20.44, and 8.29, 9.38, 8.86, and 9.60 in men and women, respectively. Compared with quartile 1, Cox proportional hazard regression analysis showed that the other three RHR groups had an increased risk of NOD after adjusting for age, gender, systolic blood pressure, diastolic blood pressure, and other risk factors. The hazard ratio values for these groups were 1.20 (95%CI:1.04-1.40, P < 0.05), 1.25 (95%CI:1.07-1.45, P < 0.01) and 1.58 (95%CI:1.36-1.82, P < 0.01), respectively. Furthermore, after adjusted the FBG, risk of NOD was significantly higher in quartile 2 (HR = 1.21, 95%CI:1.04-1.40, P < 0.01) and quartile 4 (HR = 1.22, 95%CI:1.06-1.41, P < 0.01 compared that in quartile 1. After adjusting for the factors listed above, the influence of RHR on NOD was not significant in women (P > 0.05) , but there was still an increased risk of NOD in men compared with quartile 1 with hazard ratio values of 1.21 (95%CI:1.02-1.43, P < 0.05) , and 1.27 (95%CI:1.09-1.49, P < 0.01) for quartile 2 and quartile 4, respectively. CONCLUSION: Higher RHR is linked with higher risk of NOD in population without hypertension.

Construction of the tissue engineering seed cell (HaCaT-EGF) and analysis of its biological characteristics.

OBJECTIVE: To construct the tissue engineering seed cell (HaCaT cell line) with stable expression of the human epidermal growth factor (EGF), and analyze the changes of its biological characteristics. METHODS: PCDNA3.1-EGF eukaryotic expression vector was transferred into HaCaT cell, and G418 was utilized to select the HaCaT-EGF cell line. Using an inverted microscope, PCR, ELISA method to detect the changes of the cell morphology, the expression of the EGF gene and protein, and the mRNA expression levels of apoptosis related molecule Caspase-3, the cell cycle related protein cyclin D1. RESULTS: The mRNA expression levels of the obtained HaCaT-EGF cell were more than 100 times higher than the level of ordinary HaCaT cell. The colony of the HaCaT-EGF cells was more focused and tight compared to the empty vector transfected HaCaT cells and normal HaCaT cells. The expression levels of apoptotic factor Caspase-3 and cyclin D1 in HaCaT-EGF cell were significantly higher than those in the empty vector HaCaT- pcDNA3.1 cell, and the differences were statistically significant (P<0.01), but there was no significant difference compared to the normal HaCaT cells (P>0.05). CONCLUSIONS: HaCaT-EGF cell can continuously secrete EGF, and the biological characteristic is stable. It can be used for tissue engineering experiment and is an ideal seed cell for constructing tissue engineered skin.

[Effects of adipose-derived stem cells on renal injury in burn mice with sepsis].

OBJECTIVE: To investigate the effect of adipose-derived stem cells (ADSC) on renal injury in mice with burn injury and sepsis and its underlying mechanism. METHODS: (1) Adipose tissue was collected from both inguinal regions of 5 C57BL/6J mice to isolate, culture and purify ADSC through enzyme digestion, density gradient centrifugation, and adherence method. Cells of the third passage were used in the experiment. The morphologic change in cells was observed and the growth curve of cells was determined. The expression of cell surface antigen phenotype was analyzed by flow cytometry, and the cells were identified by adipogenic and osteogenic differentiation. (2) Another 37 C57BL/6J mice were divided into normal control group (n = 5), saline group (n = 16), and group ADSC (n = 16) according to the random number table. The mice in saline group and group ADSC were injected with Pseudomonas aeruginosa after being subjected to 15% TBSA full-thickness burn on the back to reproduce septic burn model. Then the mice were injected with saline and ADSC through tail vein respectively. At post burn hour (PBH) 12, 24, 48, and 72, the pathological change in kidney tissue was observed, the levels of blood urea nitrogen and serum creatinine were determined, and the levels of TNF-α, IL-12, IL-10, and cyclooxygenase-2 (COX2) mRNA were determined with real-time fluorescence quantitative PCR in both groups. Above-mentioned indexes were also examined in the normal control group (without burn). Data were processed with multifactor analysis of variance and LSD- t test. RESULTS: (1) Cells in the third passage were orderly arranged with the shape similar to fibroblasts. The percentages of CD90(+), CD105(+), CD34(-), and CD45(-) cells were all above 90%. The cells could differentiate into osteoblasts and adipocytes. The cells were identified to be ADSC. (2) From PBH 12 to PBH 72, the neutrophil infiltration gradually increased, and the structure of kidney tubules and glomeruli were deranged in saline group. The pathological change in kidney tissue in group ADSC was less serious than that of normal control group at each time point. From PBH 12 to PBH 72, the levels of blood urea nitrogen and serum creatinine in saline group were significantly higher than those of normal control group and group ADSC (P values all below 0.01). Compared with those of the normal control group, the levels of TNF-α and IL-12 mRNA were higher in group ADSC and saline group at PBH 24 (P values all below 0.05). At PBH 24, the level of TNF-α mRNA in group ADSC (1.58 ± 0.19) was lower than that of saline group (3.36 ± 0.30, P < 0.05). At PBH 24, the levels of IL-10 and COX2 mRNA in group ADSC (2.89 ± 0.47, 4.90 ± 0.59) were higher than those in normal control group (1.00 ± 0.15, 1.00 ± 0.27) and saline group (1.32 ± 0.38, 1.57 ± 0.38, P values all below 0.05). CONCLUSIONS: ADSC can decrease the levels of blood urea nitrogen and serum creatinine, promote the production of anti-inflammatory cytokines IL-10 and COX2, and reduce the release of the pro-inflammatory cytokines TNF-α and IL-12 to offer protective effects against renal injury in burn mice with sepsis.

Protection against TGF-ß1-induced fibrosis effects of IL-10 on dermal fibroblasts and its potential therapeutics for the reduction of skin scarring.

Scarring, tightly associated with fibrosis, is a significant symptomatic clinical problem. Interleukin 10 (IL-10) has been identified as a candidate scar-improving therapy based on preclinical studies. However, the molecular mechanism of IL-10 in scar improvement is still uncertain. In this study, human dermal fibroblasts stimulated with TGF-ß1 were treated with IL-10 to analyze the mRNA and some of proteins' expression levels of type I collagen (Col1), type III collagen (Col3), alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-1 (MMP1), MMP2, MMP8 and tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2 by real-time PCR and Western blot, to observe α-SMA-positive fibroblasts by immunocytochemistry. The contracture and improvement of fibroblast-populated collagen lattice (FPCL) and a murine model of wound healing were used to evaluate the scar-improving effects by histological staining. The results showed that IL-10 can significantly down-regulate the mRNA and protein expression levels of Col1, Col3, α-SMA, and up-regulate the mRNA expression levels of MMP1 and MMP8, and decrease α-SMA-positive fibroblasts. FPCL analysis showed that the IL-10 (20 ng/ml) can significantly inhibit the contracture, improve the architecture of FPCL. Wounds injected with IL-10 demonstrated that the appearance of scar was improved, the wound margin of scarring was narrow, and the deposition of collagens (Col1 and Col3) in regenerated tissue was relieved. These results provide direct evidences that IL-10 has the inhibitory effects on the excessive deposition of extracellular matrix components and fibroblast-to-myofibroblast transition, and show that IL-10 has the potential therapy in prevention and reduction of skin scarring.

[Effects of Angelica dahurica extract on biological behavior of dermal fibroblasts].

OBJECTIVE: To observe the effects of Angelica dahurica extracts on the biological characteristics of human dermal fibroblasts in vitro and to preliminary explore its possible therapeutic mechanism for wound healing. METHODS: The optimal concentration of Angelica dahurica extracts was identified by analysing of proliferation activity of human normal fibroblasts (Fb) that treated with different concentration of Angelica dahurica extracts through thiazole blue (MTT) colorimetric assay. Cell cycle, collagen I and collagen III mRNA levels of the optimal Angelica dahurica extracts treated Fb were detected by flow cytometry (FCM) and real-time PCR techniques. RESULTS: At concentrations of 5 × 10(-4) to 5 × 10(-2) g/L, the Angelica dahurica extracts significantly enhanced the proliferation of Fb. The most significant concentration was 5 × 10(-3) g/L (t = 5.79, P < 0.01), at which an increased percentage of G1 to S and S to G2 phase cells (t = 11.2, 5.69, 2.44, P < 0.05) as well as an increased level of collagen I (1.61 ± 0.26 vs. 1.00 ± 0.16) and collagen III mRNA (3.36 ± 0.40 vs. 1.00 ± 0.14) were obtained compared to the control group (t = 6.69, 7.64, P < 0.01). CONCLUSIONS: Angelica dahurica extracts can notably promote the proliferation of Fb and accelerating the cell cycle of Fb as well as up-regulating the expression of collagen I and collagen III, which may enhance the process of wound healing.

[Study on the effects of different risk stratifications in patients with diabetes mellitus: a clinical endpoint event].

OBJECTIVE: To estimate the relationship between the risk stratification of patients with diabetes and their clinical endpoint events. METHODS: In this cohort study, we prospectively followed 8302 individuals under the following situations:contents of fasting plasma glucose ≥ 7.0 mmol/L, being diagnosed as diabetes or having used hypoglycemic drugs from Kailuan study in which 101 510 employees (81 110 males, 20 400 females, who were being employed and those retired from the company were included) from the Kailuan Company, were screened. During the 38 - 53 (48.01 ± 3.14) months of follow-up period, a new heart or cerebrovascular events were ascertained every six months. The impacts of different risk stratification in diabetic population on the incidence rates of cardiovascular and cerebrovascular events were estimated. RESULTS: Using the definitions of "people with ischemic cardiovascular disease incidence of 10-year risk assessment methods" developed by the Chinese Academy of Medical Sciences, Institute of Cardiovascular Disease, the study cohort was divided into four groups, namely, very low-risk, low risk, medium risk and high risk. (1) Along with the increasing risk of the disease, the incidence rates of total cardiovascular and cerebrovascular events, myocardial infarction, stroke, cardiovascular death and all-cause death rate also gradually increased and the differences were statistically significant (P < 0.01). However, the difference on incidence rate of sudden death was not significantly different (P > 0.05). (2) Compared to the very low-risk group, the age and sex adjusted relative risk for cardiovascular and cerebrovascular events were 1.42 (95%CI: 1.02 - 1.96, P < 0.05), 2.26 (95%CI: 1.67 - 3.04, P < 0.01) for those with medium and high risk groups, respectively. CONCLUSION: In diabetic patients, those risk factors as age, hypertension, body mass index, total cholesterol and smoking having been used on ischemic cardiovascular disease, could also be used to predict the occurrence of cardiovascular events. Along with the increasing risk factors, the risk of cardiovascular events incidence also increased.

[The prevalence and distribution of ideal cardiovascular health behavior in employees of Kailuan Group cohort].

OBJECTIVE: To observe the prevalence and distribution of ideal cardiovascular health behavior. METHODS: Health examination data between 2008 to 2009 from the employees of Kailuan Group were analyzed. RESULTS: A total of 101 333 subjects took part in the health examination, subjects with previous myocardial infarction (n = 871), stroke (n = 2255), myocardial infarction and stroke (n = 162) and subjects with incomplete examination data (n = 9311) were excluded and 88 534 subjects were included for final analysis [mean age (50.6 ± 12.3) years, male 69 916]. (1) Body mass index (BMI), systolic and diastolic pressure, cholesterol (TC) and triglyceride were significantly higher in males than in females (all P < 0.05), women's income and the education lever were significantly higher than men (P < 0.05). (2) The distribution of ideal cardiovascular health behavior (smoking, BMI, physical exercise, salt intake) was 55.8%, 41.4%, 18.9% and 14.0% respectively among the population; the ideal cardiovascular factors (fasting blood glucose, TC, blood pressure) was 80.9%, 61.8% and 18.5%, respectively. (3) The subjects with distribution of seven, six, five, four ideal cardiovascular health behavior and factors was 0.1%, 1.9%, 9.1%, 20.3%, respectively. (4) Multiple logistic regression analysis showed that female, age < 55 and high education level were associated with the ideal cardiovascular health status with a RR value (95%CI) of 4.52 (4.32 - 4.72), 1.46 (1.39 - 1.53) and 2.23 (2.10 - 2.37), respectively. CONCLUSION: The prevalence of ideal cardiovascular health is extremely low in the study population, most persons were not in the ideal cardiovascular health behavior and factors and female, age < 55 and high education level are linked with ideal cardiovascular health status.

A potential skin substitute constructed with hEGF gene modified HaCaT cells for treatment of burn wounds in a rat model.

This study aimed to investigate the feasibility of using an immortal keratinocyte cell line, HaCaT cells, to effectively deliver epidermal growth factor (EGF) in a skin substitute to treat burn wounds. The skin equivalent was constructed with human EGF (hEGF) gene modified HaCaT cells obtained through stable gene transfection; these were applied to full thickness burn wounds in a rat model. The results showed that the hEGF gene modified HaCaT cells produced more than 390ng/l of bioactive hEGF in the culture supernatant. K19 and integrin-ß1 as keratinocyte differentiation markers were elevated in the hEGF gene modified HaCaT cells which were shown to be non-tumorigenic. The skin equivalent constructed with hEGF gene modified HaCaT cells demonstrated improved epidermal morphogenesis with a thick and compact epidermis. Wound healing was accelerated noticeably when applied with this skin substitute seeded with hEGF gene modified HaCaT cells in vivo. The results suggest that HaCaT cells modified with hEGF gene might be promising seed cells for construction of genetically modified skin substitute which can effectively secrete hEGF to accelerate wound repair and regeneration.

Reduced expression of microtubule-associated protein 1 light chain 3 in hypertrophic scars.

Autophagy is a tightly regulated physiological process essential for cellular maintenance, differentiation, development, and homeostasis. Aberration of this process associates with the pathogeneses of several diseases in mammals. Hypertrophic scar (HS) is characterized by an abundance of collagenous tissue with hypercellularity. However, the molecular mechanism in HS formation is poorly understood. We compared the autophagic capacity in HS and its normal skin (NS) counterparts and explored the molecular mechanism of autophagy during the formation of HS. Microtubule-associated protein 1 light chain 3 (LC3) proteins in HS and NS were detected by immunohistochemistry, Western blot and quantitative real-time PCR (qPCR). The data showed that LC3 positive staining in HS was less intensive relative to NS group (p < 0.05). Three forms of LC3, with molecular weights of about 19 kDa (proLC3), 18 kDa (LC3-I) and 16 kDa (LC3-II), respectively, expressed in NS by Western blot. In contrast, only proLC3 expressed while both LC3-I and LC3-II were significantly downregulated in HS. The protein level of beclin 1 in HS was significantly lower compared with NS (p < 0.05). LC3 and beclin 1 mRNA levels in HS were significantly lower than that in NS (p < 0.05). These results suggest that the generation of LC3-I and LC3-II are interrupted in HS, and that the resultant decrease of autophagic capacity may associate with the pathogenesis of HS.

[Different expression of protein in the supernatant of heat injured keratinocytes].

OBJECTIVE: To compare the difference of protein expression in the supernatant of heat injured keratinocytes (KC) and normal KC. METHODS: A model of heat injured KC was produced in vitro. The supernatant of normal KC and heat injured KC was collected after culture for 12 hours, and was ultrafiltered and lyophilized to get the protein. The protein sample was separated by immobilized pH gradient based two dimensional gel electrophoresis (2-DE). The gel was stained and the different expression of protein was analyzed using ImageMaster 2D analysis software. RESULTS: (1) Average protein spots were 1,898 +/- 113, 1,877 +/- 97 in the supernatant of normal and heat injured KC and 1,118 protein spots could be used for statistical analysis. (2) Statistical result showed that 26 protein spots were significantly different between the two groups. 16 protein spots were higher in the supernatant of normal KC and then 10 protein spots were lower in the normal group. (3) 16 protein spots, which included 10 kinds of proteins, were identified successfully as different spots. Lower expression proteins were alpha-enolase, actin cytoplasmic 2, peroxiredoxin-4, phosphoglycerate mutase 1, G protein-regulated inducer of neurite outgrowth l in the supernatant of heat injured KC. Higher expression proteins in heat KC were purine nucleoside phosphorylase, tumor necrosis factor ligand superfamily member 10, proteasome subunit alpha type-7, UDP-glucose 6-dehydrogenase in the supernatant of heat injured KC. CONCLUSIONS: The result indicated that there are some significant different expression proteins in the supernatant of normal KC and heat injured KC. These findings provide new data for screening major molecules of tissue repair and finding the mechanism of wound repair.

[Variation and clinical significance of serum CTnT and CK-MB in patients with acute organophosphorus pesticid poisoning].

AIM: to explore the variation and clinical significance of serum CTnT and CK-MB in patients with acute organophosphorus pesticid poisoning (AOPP). METHODS: 100 cases of patients with AOPP(AOPP-group) and 100 healthy subjects were studied, the serum CTnT and CK-MB level were detected using ELISA and immunosuppression methods. RESULTS: after poisoning 6 h, 24h, 72h, the serum CTnT and CK-MB levels of the AOPP group were higher than the controls group, compared difference was significant (P<0.05); The serum CTnT and CK-MB levels increased with increasing degrees of poisoning, the different degrees of poisoning was positively correlated with CTnT and CK-MB levels (r=0.569, 0.498, P<0.01). CONCLUSION: combined monitoring of serum CTnT and CK-MB of AOPP patients can help determine the extend of poisoning, when the serum CTnT and CK-MB levels of the AOPP group increased, the degrees of heart damage is serious, which is important diagnostic significance for heart damage.

Smad interacting protein 1 as a regulator of skin fibrosis in pathological scars.

Keloids and hypertrophic scars are significant symptomatic clinical problems characterized by the excessive and abnormal deposition of collagen-based extracellular matrix (ECM) components. However, the molecular basis of keloid and hypertrophic scar formation has not been fully elucidated. Here, we demonstrated that down-regulation of the transcription factor Smad interacting protein 1 (SIP1) could be relevant to keloid and hypertrophic scar formation. The results of the present study show that the level of SIP1 mRNA is significantly decreased in pathological scar tissues and in normal skin and pathological scar fibroblasts treated with transforming growth factor ß1 (TGF-ß1). In contrast, the expression of SIP1 mRNA is not decreased in normotrophic scar samples. The SIP1 mRNA level inversely correlates with the mRNA level of type I collagen (COL1A2) and directly correlates with the mRNA level of matrix metalloproteinase-1 (MMP1). Overexpression of SIP1 in keloid and hypertrophic scar fibroblasts represses TGF-ß1-stimulated COL1A2 expression and induces MMP1 expression. Alternatively, knockdown of SIP1 in normal skin fibroblasts enhance TGF-ß1-induced COL1A2 levels. These findings suggest that SIP1 could be a regulator of skin fibrosis, and depletion of SIP1 in pathological scar tissues could result in an up-regulation of collagen and down-regulation of matrix metalloproteinase, leading to an abnormal accumulation of ECM along with fibrosis and pathological scar formation.

[Effect of heat injured keratinocytes supernatant on biological behavior of fibroblasts].

OBJECTIVE: To observe the effect of the supernatant of heat injured keratinocytes (KC) on biological behavior of the dermal fibroblasts (Fb). METHODS: Human dermal Fb were isolated and cultured. A model of heat injured KC (HaCaT) was reproduced in vitro. Supernatant of normal KC and the supernatant of KC culture 12 hours after heat injury were collected and diluted with non-serum DMEM in 1:1 volume ratio to make normal KC conditioned medium (NKCM) and heat injury KC conditioned medium (HKCM) respectively. Fb was respectively treated with non-serum DMEM and 2 kinds of conditioned medium. (1) The proliferation of Fb was detected with MTT method at post culture hour (PCH) 12, 24, 36, 48. (2) The apoptosis of Fb was determined by flow cytometry at PCH 12 (Fb were heat injured in advance; Fb without heat treatment was used as control). (3) At PCH 24, expression of a-SMA in Fb cytoplasm was determined with immunofluorescence method; expression of a-SMA mRNA in Fb was determined with real-time quantified PCR. Data were processed with one-way analysis of variance, and pairwise comparison among groups with LSD-t test. RESULTS: (1) The proliferation of Fb: the absorbance value of Fb cultured with HKCM at PCH 12, 24, 36, 48 was respectively higher than that of Fb cultured with non-serum DMEM (with t value respectively 1.89, 2.35, 2.02, 1.94, and P values all below 0.01). There were significant statistical differences between the absorbance values of Fb cultured with HKCM and those of Fb cultured with NKCM at PCH 12, 24, and 48 (at PCH 12, t = 1.83, P < 0.01; at PCH 24, t = 2.91, P < 0.05; at PCH 48, t = 1.83, P < 0.05). (2) Apoptosis of Fb cultured with HKCM was diminished as compared with that of Fb cultured with NKCM and of Fb without treatment (t = 3.31, P < 0.05; t = 1.47, P < 0.01). (3) The expression of alpha-SMA (red fluorescence) in Fb cultured with non-serum DMEM or NKCM was less as seen under fluorescence scope, and it was obviously increased in Fb cultured with HKCM. (4) The relative expression amount of alpha-SMA mRNA in Fb cultured with HKCM was 1.32 +/- 0.06, which was higher than that both in Fb cultured with NKCM (1.14 +/- 0.07, t = 2.51, P < 0.05) and in Fb cultured with non-serum DMEM (1.00 +/- 0.09, t = 1.77, P < 0.05). CONCLUSIONS: The supernatant of KC 12 hours after heat injury can obviously promote the proliferation of Fb, inhibit its apoptosis and accelerate transdifferentiation of Fb to myofibroblasts.