Annexin A1-derived peptide Ac2-26 facilitates wound healing in diabetic mice.

Impaired wound healing is a common complication of diabetes. In diabetic wounds, macrophages present dysfunctional efferocytosis and abnormal phenotypes, which could result in excessive neutrophil accumulation and prolonged inflammation, thereby eventually hindering wound repair. ANXA1 N-terminal peptide Ac2-26 exhibits a high potential in mitigating inflammation and improving repair; however, its efficacy in diabetic wound repair remains unclear. In this study, a cutaneous excisional wound model was built in genetically diabetic mice. Ac2-26 or a vehicle solution was employed locally in wound sites. Subsequently, wound zones were measured and sampled at different time intervals post-wounding. Using hematoxylin-eosin and Masson's trichrome staining, we observed the histopathological variations and collagen deposition in wound samples. Based on immunohistochemistry and immunofluorescence, the numbers of neutrophils, macrophages, and CD206-positive macrophages in the wound samples were determined. Cytokine expression in wound samples was studied by immunoblot assay. Results showed that Ac2-26 treatment could facilitate diabetic wound closure, down-regulate the number of neutrophils, and improve angiogenesis and collagen deposition. In addition, Ac2-26 application expedited macrophage recruitment and up-regulated the percentage of macrophages expressing CD206, which is a marker for M2 macrophages. Moreover, Ac2-26 inhibited the expressions of TNF-α and IL-6 and up-regulated the expressions of IL-10, TGF-ß, and VEGFA during diabetic wound healing. Hence, based on the aforementioned findings, Ac2-26 application in diabetic wounds could exert anti-inflammatory and pro-repair effects by reducing neutrophil accumulation and facilitating M2 macrophage development.

Detection of RAGE expression and its application to diabetic wound age estimation.

With the prevalence of diabetes, it is becoming important to analyze the diabetic wound age in forensic practice. The present study investigated the time-dependent expression of receptor for advanced glycation end products (RAGE) during diabetic wound healing in mice and its applicability to wound age determination by immunohistochemistry, double immunofluorescence, and Western blotting. After an incision was created in genetically diabetic db/db mice and control mice, mice were killed at posttraumatic intervals ranging from 6 h to 14 days, followed by the sampling of wound margin. Compared with control mice, diabetic mice showed the delayed wound healing. In control and diabetic wound specimens, RAGE immunoreactivity was observed in a small number of polymorphonuclear cells (PMNs), a number of macrophages, and fibroblasts. Morphometrically, the positive ratios of RAGE in macrophages or fibroblasts considerably increased in diabetic wounds during late repair, which exceeded 60% at 7 and 10 days post-injury. There were no control wound specimens to show a ratio of >60% in macrophages or fibroblasts. By Western blotting analysis, the ratios of RAGE to GAPDH were >1.4 in all diabetic wound samples from 7 to 10 days post-injury, which were >1.8 at 10 days after injury. By comparison, no control wound specimens indicated a ratio of >1.4. In conclusion, the expression of RAGE is upregulated and temporally distributed in macrophages and fibroblasts during diabetic wound healing, which might be closely involved in prolonged inflammation and deficient healing. Moreover, RAGE is promising as a useful marker for diabetic wound age determination.

The time-dependent expression of α7nAChR during skeletal muscle wound healing in rats.

The study on time-dependent expression of α7 nicotine acetylcholine receptor (α7nAChR) was performed by immunohistochemistry, Western blotting, and real-time PCR during skeletal muscle wound healing in rats. Furthermore, co-localization of α7nAChR with macrophage or myofibroblast marker was detected by double immunofluorescence. A total of 50 Sprague-Dawley male rats were divided into control and contusion groups (3 h, 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury). In the uninjured controls, α7nAChR positive staining was observed in the sarcolemma and sarcoplasm of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells, a number of macrophages and myofibroblasts showed positive reaction for α7nAChR in contused zones. Morphometrically, the average ratios of α7nAChR-positive cells were over 50 % from 3 to 10 days after contusion, and exceeded 60 % at 5 and 7 days post-injury. Besides, the positive ratios of α7nAChR were <50 % at the other posttraumatic intervals. By Western blotting analysis, the average ratio of α7nAChR protein expression maximized at 7 days after injury, which was >2.13. Similarly, the relative quantity of α7nAChR mRNA expression peaked at 7 days post-wounding as compared with control by real-time PCR detection, showing a relative quantity of >2.65. In conclusion, the expression of α7nAChR is upregulated and temporally distributed in macrophages and myofibroblasts during skeletal muscle wound healing, which might be closely involved in inflammatory response and fibrotic repair after injury. Moreover, α7nAChR is promising as a useful marker for wound age determination of skeletal muscle.

Activation of α7nAChR Promotes Diabetic Wound Healing by Suppressing AGE-Induced TNF-α Production.

Diabetes frequently presents accumulation of advanced glycation end products (AGEs), which might induce excessive TNF-α production from macrophages to cause impaired wound healing. Recent studies have shown that activation of α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages efficiently suppressed TNF-α synthesis. The aim of this study was to investigate the accumulation of AGEs in the wounds and determine whether PNU282987, an α7nAChR agonist, can improve wound repair by inhibiting AGE-mediated TNF-α production in a streptozotocin (STZ)-induced diabetic mouse model. Animals were assigned into four groups: wounded control group, wounded diabetic group, wounded diabetic group treated intraperitoneally with PNU282987, or wounded diabetic group treated intraperitoneally with vehicle. Compared with the non-diabetic control mice, the diabetic mice exhibited delayed wound healing that was characterized by elevated accumulation of AGEs, increased TNF-α level and macrophage infiltration, and decreased fibroblast number and collagen deposition at the late stage of repair. Besides, macrophages of diabetic wounds showed expression of α7nAChR. During late repair, PNU282987 treatment of diabetic mice significantly reduced the level of TNF-α, accelerated wound healing, and elevated fibroblast number and collagen deposition. To investigate the cellular mechanism of these observations, RAW 264.7 cells, a macrophage cell line, were incubated with AGEs in the presence or absence of PNU282987. TNF-α production from AGE-stimulated macrophages was significantly decreased by PNU282987 in a dose-dependent manner. Furthermore, PNU282987 significantly inhibited AGE-induced nuclear factor-κB (NF-κB) activation and receptor for AGE (RAGE) expression. These results strongly suggest that activating α7nAChR can promote diabetic wound healing by suppressing AGE-induced TNF-α production, which may be closely associated with the blockage of NF-κB activation in macrophages.

Human cytomegalovirus-encoded US28 may act as a tumor promoter in colorectal cancer.

AIM: To assess human cytomegalovirus-encoded US28 gene function in colorectal cancer (CRC) pathogenesis. METHODS: Immunohistochemical analysis was performed to determine US28 expression in 103 CRC patient samples and 98 corresponding adjacent noncancerous samples. Patient data were compared by age, sex, tumor location, histological grade, Dukes' stage, and overall mean survival time. In addition, the US28 gene was transiently transfected into the CRC LOVO cell line, and cell proliferation was assessed using a cell counting kit-8 assay. Cell cycle analysis by flow cytometry and a cell invasion transwell assay were also carried out. RESULTS: US28 levels were clearly higher in CRC tissues (38.8%) than in adjacent noncancerous samples (7.1%) (P = 0.000). Interestingly, elevated US28 amounts in CRC tissues were significantly associated with histological grade, metastasis, Dukes' stage, and overall survival (all P < 0.05); meanwhile, US28 expression was not significantly correlated with age, sex or tumor location. In addition, multivariate Cox regression data revealed US28 level as an independent CRC prognostic marker (P = 0.000). LOVO cells successfully transfected with the US28 gene exhibited higher viability, greater chemotherapy resistance, accelerated cell cycle progression, and increased invasion ability. CONCLUSION: US28 expression is predictive of poor prognosis and may promote CRC.

Analysis of Forensic Autopsy in 120 Cases of Medical Disputes Among Different Levels of Institutional Settings.

Despite advances in medical science, the causes of death can sometimes only be determined by pathologists after a complete autopsy. Few studies have investigated the importance of forensic autopsy in medically disputed cases among different levels of institutional settings. Our study aimed to analyze forensic autopsy in 120 cases of medical disputes among five levels of institutional settings between 2001 and 2012 in Wenzhou, China. The results showed an overall concordance rate of 55%. Of the 39% of clinically missed diagnosis, cardiovascular pathology comprises 55.32%, while respiratory pathology accounts for the remaining 44. 68%. Factors that increase the likelihood of missed diagnoses were private clinics, community settings, and county hospitals. These results support that autopsy remains an important tool in establishing causes of death in medically disputed case, which may directly determine or exclude the fault of medical care and therefore in helping in resolving these cases.

[The effects of Kangai injection on enzyme activities of macrophages in rats].

OBJECTIVE: To study the effects of Kangai injection on the enzyme activities of macrophages and morphology of spleen and thymus from rats. METHODS: Twenty four male SD rats were randomly divided into two groups (n = 12), normal control group and experimental group. The rats in experimental group were injected with Kangai injection at the dosage of 5 ml/kg x d for 30 days peritoneally and those in control group were injected with nomal saline at the same volume. The content of supermicro protein was assayed by BCA method, the activities of lactate dehydrogenase (LDH), glutathione peroxidase(GSH-Px), and inducible nitric oxide synthase (iNOS) from alveolar macrophages(AM) and peritoneal macrophages (PM) were detected biochemically. The activities of acid phosphatase (ACP), superoxide dismutase(SOD) and succinate dehydrogenase (SDH) from AM and PM were detected by ELISA. The morphology of spleen and thymus were observed by light microscopy. RESULTS: The activities of LDH, GSH-Px and iNOS within AM and PM from experimental group were increased significantly compared with those of control group (P < 0.05). The activities of ACP, SOD and SDH in AM and PM from experimental group were also higher than those from control group (P < 0.05). Microscopically, there was thickening of peripheral arterial lymphatic sheath, enlargement of splenic lymphoid nodules with expended germinal center in the spleen of experimental group. There was no significant difference in the mophology of thymus between the two groups. CONCLUSION: Kangai injection may improve immune function by activating macrophages.

[Study on the mechanism of how curcumin improves pulmonary vascular remodeling associated with chronic pulmonary arterial hypertension].

OBJECTIVE: To investigate the mechanism of how curcumin improves pulmonary vascular remodeling associated with chronic pulmonary arterial hypertension. METHODS: The model of chromic hypoxia hypercapniapulmoary remodeling was made. Twenty-four male rats were randomly divided into 4 groups (n = 6): group I (normoxia control group), group II (hypxia and hypercapnia model group), group II (disodium cromoglycate control group), group IV (curcumin treated group). The last 3 group rats were put in a hypoxia cabin where the concentrate of O2 was 8% - 11% and the concentrate of CO2 was 3% - 5%, for 8 h a day and lasting 4 w in total. Group III rats were intraperitoneally injected with disodium cromoglycate (20 mg/kg) and group IV rats were administrated with curcumin by gavage (150 mg/kg). The morphological changes of pulmonary vessel walls and the ultrastructure of mast cells were observed by the optics microscope and the transmission electron microscope. Mast cells and its degranulation state were measured by toluidine blue staining and immunohistochemistry. Data were expressed as means ± SD (standard deviation) and analyzed with SPSS17.0 software. RESULTS: (1) By optics microscopy observation, the value of WA/TA was significantly higher in II group than other groups (P < 0.05). (2) Electron microscope showed that the endothelial cells of pulmonary arterioles in III and IV group were near to I group and the proliferation of pulmonary arterial media smooth cell layer and collagen fibers in adventitia was much lighter than those in II group. The membrane of mast cells was more intact in I, III, IV group than II group. (3) The number of mast cells, the degranulation rate of master cells and the number of positive tryptase stained cells in II group were significantly more than those in other groups. (P < 0.05). CONCLUSION: Curcumin may inhibit the remodeling of pulmonary vessel induced by chronic hypoxia hypercapnia by mast cell regulation.

Time-dependent expression and distribution of Egr-1 during skeletal muscle wound healing in rats.

Recent studies have shown that early growth response factor-1 (Egr-1) plays an important role in regulation of inflammation and tissue repair, but little is known about its expression after trauma to skeletal muscles. A preliminary study on time-dependent expression and distribution of Egr-1 was performed by immunohistochemistry, immunofluorescence and Western blotting during skeletal muscle wound healing in rats. An animal model of skeletal muscle contusion was established in 45 Sprague-Dawley male rats. Samples were taken at 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days and 21 days post-injury, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. In the uninjured controls, Egr-1 positive staining was observed in the sarcoplasm and nuclei of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells (PMNs), a number of mononuclear cells (MNCs), fibroblastic cells (FBCs) and regenerated multinucleated myotubes showed positive reaction for Egr-1 in contused zones. By morphometric analysis, an increase in Egr-1 expression was verified at inflammatory phase after contusion, which reached a peak in the regenerated phase overlapping with the fibrotic phase during skeletal muscle wound healing. The expression tendency was further confirmed by Western blotting assay. By immunofluorescent staining for co-localization, the Egr-1-positive MNCs and FBCs in wounds were identified as macrophages and myofibroblasts. The results demonstrate that the expression of Egr-1 is up-regulated and temporally distributed in certain cell types after trauma to skeletal muscles, which may be closely involved in inflammatory response, fibrotic repair and muscle regeneration during skeletal muscle wound healing.