Objective To prepare nerve growth factor (NGF)-insulin composite gel and observe the effects of NGF-insulin composite gel on deep second degree scald wound healing in diabetic rats. Methods Carbomer 980, NGF (4 000 U), and insulin (800 U) were used to prepare the insulin gel, NGF gel, and NGF-insulin composite gel. The character of NGF-insulin composite gel was observed, and the in vitro drug release was tested. Seventy-five SPF Wistar male rats, weighing 200-250 g, were divided into 5 groups randomly, 15 rats each group: normal control group (group A), diabetes control group (group B), insulin gel treatment group (group C), NGF gel treatment group (group D), and NGF-insulin composite gel treatment group (group E). The type 1 diabetes rat model was established by intraperitoneal injection of Streptozotocin (55 mg/kg) in groups B, C, D, and E, while the rats in group A were injected with the same dose of citric acid and calcium citrate buffer. After modeling success, deep second degree scald wound on the back was made with constant temperature water bath box. Wounds were treated with carbomer blank gel in groups A and B, with insulin composite gel in group C, with NGF gel in group D, and with NGF-insulin composite gel in group E, once a day. At 3, 7, 11, 15, and 21 days after injury, the scald wound healing was observed and healing rate was calculated; the full-thickness skin specimens were harvested from 3 rats of each group for histological and immuohistochemical staining observation. Results The NGF-insulin composite gel was clear and transparent, and had good moisture retention capacity and adhesion; it was easy to apply and clean up. The drug release in vitro lasted more than 24 hours and maintained for 30 days. No rat died during the experiment. At 3 days after injury, wound area did not reduce in all groups; at 7, 11, 15, and 21 days, group E had the highest wound healing rate, and group B had the lowest; significant differences were found between group E and group B and when compared with the other groups (P lt; 0.05). HE staining showed that group E surpassed other groups in the growth of granulation tissue and collagen fiber. Immunohistochemical results showed that the CD34 and proliferating cell nuclear antigen (PCNA) expressed at 3 days, and the number of positive cells increased gradually with time; the microvessel density and PCNA expression were highest in group E and were lowest in group B, showing significant differences when compared with the other groups and between group E and group B (P lt; 0.05). Conclusion NGF-insulin composite gel can improve deep second degree scald wound healing in diabetic rats.
Objective To explore the possible mechanism of nerve growth factor (NGF) mixed insul in on the angiogenesis of burn wounds and the effect on the expressions of Bcl-2 and Bax in diabetic rats. Methods A total of 75 SPF male Wistar rats, weighing 200-220 g, were selected randomly and divided into nomal control (group A, n=15), the rats with diabetic control (group B, n=15), insul in treatment (group C, n=15), NGF treatment (group D, n=15), NGF and insul in treatment (group E, n=15) groups. In groups B, C, D, and E, streptozotocin was given by intraperitoneal injection at dose of 10 mg/kg on the 1st day and 50 mg/kg on the 3rd day to prepare the diabetic rat models. In group A, citric acid buffer at the samedose was given. After 1 month of diabetic models, second degree scald was made on the back of the rats, and then wounds were treated with 3-layer normal sal ine gauze in groups A and B, with 3-layer gauze containing 5 U Novol in 30R and subcutaneous injection of Novol in 30R (4-6 U/kg) everyday in group C, with 3-layer gauze containing 5 mL NGF (25 U/mL) in group D, and with a combination of groups C and D in group E. At 7, 11, 15, and 21 days, the wound heal ing rate was calculated; at 3, 7, 11, 15, and 21 days, the expressions of Bcl-2, Bax, and CD34 were determined and the microvascular density was measured by immunohistochemistry staining. Results All rats survived till experiment was finished. The area of wounds became smaller gradually with time. Group E was better than other groups in the wound heal ing rate (P lt; 0.05), the skin keratosis, the hair growth, and the granulation tissue and collagen fibers growth. With time, the expressions of CD34 and Bcl-2 increased gradually, reached the peak at 15 days and decreased at 21 days; the expression was ber in group E than in other groups (P lt; 0.05). At 3 days, Bax did not express; at 7 days, Bax began to express in new vascular endothel ial cells and the expression increased gradually with time; the expression was weaker in group E than in other groups (P lt; 0.05). Conclusion A combination of NGF and insul in local appl ication can enhance the angiogenesis of the burn wound in diabetic rats and accelerate wound heal ing by increasing the expression of Bcl-2 and decreasing the expression of Bax and restraining apoptosis of the wounds vascular endothel ial cells of diabetic rats.
Abstract： Objective To study the difference of gene expression profile of bone marrow mesenchymal stem cells （MSCs） cultured in vitro from coronary heart disease patient with or without diabetes mellitus by Affymetrix Gene array. Methods One male patient at age of 53 years with coronary heart disease and diabetes mellitus was included in this study with the diagnosis of coronary heart disease and type 2 diabetes mellitus. Another male patient at age of 51 years with coronary heart disease without diabetes mellitus was also included in this study with the diagnosis of coronary heart disease. MSCs of the two patients were isolated and purified by the methods of density gradient centrifugation with lymphocyte separation medium for human and adherent filtration. The MSCs expression profile of cytokines and signal transduction genes were examined by Affymetrix gene array. Results There were 27 functional protein genes expression in the patient with coronary heart disease and diabetes mellitus relating to cell apoptosis, cytokine, and signal transduction. Among them, the expression of 13 functional genes, including TNFRSF10B, TNFRSF21, NGF, CAV2, ITGA8, TNS1, ITGA2, AKT3, MBP, MAP2, INHBA, FST, PLA2G5, increased significantly in the patient with coronary heart disease and diabetes mellitus. However, the expression level of 14 genes, including EPR1, BIRC5, HELLS, BCL2, HGF, CASP1, SEPP1, ITGA9, MAP2K6, RUNX3, TGFBR2, RUNX2, CTNNB1, CDC42, decreased significantly. Conclusion The gene expression profile of bone marrow MSCs from coronary heart disease patient with diabetes mellitus is significantly different from the patient with coronary heart disease patient without diabetes mellitus.
Objective To investigate the effect of topical appl ication of insul in on the burn wound heal ing in aging diabetes mell itus (DM) rats and to explore its mechanism. Methods Seventy-five SPF Wistar rats (female and/or male), aged 12-24 months and weighing 300-350 g, were selected and randomly divided into group A (burn control group, n=25), group B(DM burn control group, n=25), and group C (DM insul in treatment group, n=25). The rats in group B and group C were fedwith high-fat, high-protein, and high-sugar forage for 1 month and received intraperitoneal injection of streptozotocin (STZ)to establ ish experimental model of aging DM. The rats were fed with high-fat, high-protein, and high-sugar forage for another 8 weeks. Then, the deep second-degree burn model was establ ished in the rats of group B and group C. The wounds in group A and B underwent local subcutaneous injection of 2 mL isotonic sal ine and group C received local subcutaneous injection of 0.1 U insul in. The rate of wound heal ing was calculated 7, 14, and 21 days after burn injury. At 1, 3, 7, 14, and 21 days after burn injury, HE staining observation, immunohistochemistry staining for CD34, detection of sugar and hydroxyprol ine (HOP) content in wound tissue, and microvessel density (MVD) calculation were performed. Results At 7, 14, and 21 days after burn injury, the wound heal ing rates of group A and group C was significantly higher than that of group B (P lt; 0.05), and there was no significant difference between group A and group C (P gt; 0.05). Histology observation at 21 days after burn injury: in group A, certain degree of epithel ization was evident in the wound epithel ium; in group B, large quantity of necrotic tissue was evident; in group C, complete epithl ization occurred in the wound epithel ium with better epithel ial cell differentiation and more neonatal collagen. For the sugar content in the wound tissue, group A was significantly lower than group B or group C at 1, 3, 7, 14, and 21 days (P lt; 0.05) and group C was significantly lower than group B at 7, 14, and 21 days (P lt; 0.05). For the HOP content in the wound tissue and the MVD count, group A or group C was significantly higher than group B (P lt; 0.05) and there was no significant difference between group A and group C (P gt; 0.05). CD34 expression: in group A, it was (+) at 7 days, (++) at 14 days, and (+++) at 21 days; in group B, it was (+) at 14 and 21 days; in group C, it was (++) at 7 days and (+++) at 14 and 21 days. Conclusion Topical appl ication of insul in can promote the synthesis of wound collagen, accelerate the woundangiogenesis, and speed up the wound heal ing in aging DM rats.
Objective To evaluate the effect of left atrial enlargement on atrial myocardial fibrosis degree and levels of the angiotensinⅡ (AngⅡ)/Rac GTPase activating protein 1 (Rac1)/signal transducersand activators of transcription 3 (STAT3) signaling pathways expressing in patients with persistent atrial fibrillation and rheumatic heart disease (RHD). Methods From March to December 2011, 30 patients with RHD who underwent prosthetic valve replacement in our hospital were enrolled, including 16 males and 14 females, aged 42-70 (56.9±6.8) years. Twenty RHD patients with persistent atrial fibrillation as a research group and ten RHD patients with sinus rhythm as a control group (group A) underwent transthoracic echocardiography and right atrial appendage (RAA) tissue samples were obtained from these patients during mitral/aortic valve replacement operation. The research group according to left atrial diameter (LAD) was divided into two groups, ten patients in each group: a group B with LAD of 50–65 mm and a group C with LAD of LAD>65 mm. For each sample, histological examination was performed by hematoxylin-eosin and Masson’s trichrome staining. Light-microscopic pictures of atrial tissues samples were stained and tissue fibrosis degree in each group was analyzed. AngⅡ concentration was measured by enzyme linked immunosorbent assay. Rac1 and STAT3 were measured by western blotting. Results LAD was significantly greater in AF patients with RHD than in the control group. Hematoxylin-eosin staining demonstrated highly organized arrangement of atrial muscles in the control group and significant derangement in both group B and group C with reduced cell density and increased cell size. Moreover, Masson’s trichrome staining showed that atrial myocytes were surrounded by large trunks of collagen fibers in both group B and group C, but not in the group A. There was a positive correlation between atrial tissue fibrosis and LAD. AngⅡ content was positively correlated with LAD. Similarly, Rac1 and STAT3 protein levels were found considerably higher in the group C and group B than in the group A with excellent correlation to LAD. Conclusion In patients with RHD complicated with persistent atrial fibrillation, the degree of atrial fibrosis and the expression level of AngⅡ/Rac1/STAT3 signaling pathways significantly increase with the left atrialenlargement.