Proliferating Cell Nuclear Antigen Positive Cells Research Articles

Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway

ObjectivePhysical exercise, a common non-drug intervention, is an important strategy in cancer treatment, including hepatocellular carcinoma (HCC). However, the mechanism remains largely unknown. Due to the importance of hypoxia and cancer stemness in the development of HCC, the present study investigated whether the anti-HCC effect of physical exercise is related to its suppression on hypoxia and cancer stemness. MethodsA physical exercise intervention of swimming (30 min/d, 5 d/week, for 4 weeks) was administered to BALB/c nude mice bearing subcutaneous human HCC tumor. The anti-HCC effect of swimming was assessed in vivo by tumor weight monitoring, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) detection of proliferating cell nuclear antigen (PCNA) and Ki67. The expression of stemness transcription factors, including Nanog homeobox (NANOG), octamer-binding transcription factor 4 (OCT-4), v-Myc avian myelocytomatosis viral oncogene homolog (C-MYC) and hypoxia-inducible factor-1α (HIF-1α), was detected using real-time reverse transcription polymerase chain reaction. A hypoxia probe was used to explore the intratumoral hypoxia status. Western blot was used to detect the expression of HIF-1α and proteins related to protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. The IHC analysis of platelet endothelial cell adhesion molecule-1 (CD31), and the immunofluorescence co-location of CD31 and desmin were used to analyze tumor blood perfusion. SMMC-7721 cells were treated with nude mice serum. The inhibition effect on cancer stemness in vitro was detected using suspension sphere experiments and the expression of stemness transcription factors. The hypoxia status was inferred by measuring the protein and mRNA levels of HIF-1α. Further, the expression of proteins related to Akt/GSK-3β/β-catenin signaling pathway was detected. ResultsSwimming significantly reduced the body weight and tumor weight in nude mice bearing HCC tumor. HE staining and IHC results showed a lower necrotic area ratio as well as fewer PCNA or Ki67 positive cells in mice receiving the swimming intervention. Swimming potently alleviated the intratumoral hypoxia, attenuated the cancer stemness, and inhibited the Akt/GSK-3β/β-catenin signaling pathway. Additionally, the desmin+/CD31+ ratio, rather than the number of CD31+ vessels, was significantly increased in swimming-treated mice. In vitro experiments showed that treating cells with the serum from the swimming intervention mice significantly reduced the formation of SMMC-7721 cell suspension sphere, as well as the mRNA expression level of stemness transcription factors. Consistent with the in vivo results, HIF-1α and Akt/GSK-3β/β-catenin signaling pathway were also inhibited in cells treated with serum from swimming group. ConclusionSwimming alleviated hypoxia and attenuated cancer stemness in HCC, through suppression of the Akt/GSK-3β/β-catenin signaling pathway. The alleviation of intratumoral hypoxia was related to the increase in blood perfusion in the tumor.Please cite this article as: Xiao CL, Zhong ZP, Lü C, Guo BJ, Chen JJ, Zhao T, Yin ZF, Li B. Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway. J Integr Med. 2023; 21(2): 184–193.

Vitamin B12 suppresses GADD153, prevents apoptosis and regulates the testicular function in methotrexate treated rat testis

ABSTRACT Methotrexate (MTX) is an anti-neoplastic drug that also causes testicular damage. Vitamin B12 (Vit B12) is a water soluble vitamin that is required for normal metabolism. We investigated Vit B12 as a possible protective agent against testicular damage caused by MTX treatment. We divided rats into four groups: control group, Vit B12 group treated with Vit B12 daily for 15 days, MTX group treated with MTX on day 8, MTX + Vit B12 group treated with MTX on day 8 + Vit B12 for 15 days. Serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone were measured. We also measured proliferating cell nuclear antigen (PCNA), connexin43 (Cx43) and the growth arrest- and DNA damage-inducible gene, 153 (GADD153), using immunohistochemical staining. Apoptosis was assessed using TUNEL staining. The MTX group exhibited degeneration of seminiferous tubules; decreased serum testosterone, LH and FSH levels; fewer PCNA positive cells; increased Cx43 expression; and increased GADD153 and TUNEL stained cells compared to the control group. These pathologic findings were substantially reversed In the MTX + Vit B12 group. MTX caused increased endoplasmic reticulum stress and apoptosis via GADD153. Consequently, Vit B12 potentially is a protective agent against damage caused by MTX.

Histopathological changes of pulmonary vascular remodeling in dogs affected with pulmonary hypertension secondary to degenerative mitral valve disease

Introduction/objectivesPulmonary hypertension (PH) can cause pulmonary arterial remodeling. Medial remodeling is a structural change of the pulmonary artery seen with PH. Hyperplasia and hypertrophy of pulmonary arterial smooth muscle cells (SMCs) are suggested as causes of medial remodeling. To demonstrate the histopathological changes of the pulmonary artery in dogs affected with PH secondary to degenerative mitral valve disease (DMVD) compared with DMVD without PH and control dogs. AnimalsLung samples obtained from the carcasses of 19 older small-breed dogs (Control, n = 5; DMVD, n = 7; DMVD + PH, n = 7). Materials and methodsLung tissue sections were stained with hematoxylin and eosin, Masson's trichrome, and proliferating cell nuclear antigen (PCNA) immunohistochemistry. ResultsThe internal diameters of the pulmonary artery in the three groups were not different. Masson's trichrome staining revealed no collagen deposition in the intimal layer of the pulmonary artery in all dogs. The external diameter, percentage of medial thickness (%MT), percentage of SMC layer and collagen deposition areas, average number of SMCs, and the percentage of PCNA positive cells (%PCNA) of the pulmonary artery were increased in the DMVD and DMVD + PH groups compared with the control group. The %PCNA in the DMVD + PH group was significantly decreased when compared with the DMVD group. ConclusionsMedial remodeling was found in left-sided heart failure DMVD dogs with and without PH. The medial remodeling in DMVD dogs with and without PH is related to SMC hyperplasia, hypertrophy, and collagen deposition, leading to an increased medial layer thickness of the pulmonary artery.

Suppression of sphingosine 1-phosphate lyase retards the liver regeneration in mice after partial hepatectomy.

Background: Liver regeneration is an extremely complicated process that is regulated by a number of signaling pathways. Sphingosine 1-phosphate (S1P), a potent bioactive lipid mediator playing crucial roles in various cellular responses through its receptors, has been attracting attention in the fields of hepatology, where S1P lyase (SPL), an irreversibly degrading enzyme of S1P, reportedly has a stimulatory role in growth of hepatocellular carcinoma (HCC).Aim of the study: To examine whether SPL might play a stimulatory role in liver regeneration.Method: Using in-vivo siRNA technology, we inhibited SPL expression. Seventy percent of the liver was resected in mice as partial hepatectomy (PH). Liver tissue samples were collected and mRNA expression level of the SPL, IHC of the proliferating cell nuclear antigen (PCNA), protein levels of various proliferation factors and lipid measurements were performed in different groups.Results: The mRNA levels of SPL increased in PH mice on the third day after PH surgery. When we suppressed the expression of SPL by in-vivo siRNA, we observed a significant decline of the PCNA positive cell numbers. Furthermore, the Cyclin D1 expressions and phosphorylation of ERK also were decreased in the siSPL injected PH group.Conclusion: We verified the importance of the SPL in liver regeneration, using the mice PH model. SPL might be a potential target to facilitate liver regeneration.

Modulating Effect of Vitamin D on iNOS, PCNA and a-SMA Expression Against Diclofenac Sodium Induced Gastric Injury in Rats

BACKGROUND: Diclofenac sodium is a nonsteroidal anti-inflammatory prescription, widely used in the management of many inflammatory diseases but the side effects limiting its clinical use. The present work was carried out to detect the ameliorative effect of vitamin D against diclofenac sodium induced gastric injury in adult male albino rats.METHODS: Forty adult male Wistar albino rats were classified into four groups: G-I received no treatment (control group), G-II orally received 500 IU/kg of vitamin D daily, G-III intraperitoneally received 3 mg/kg of diclofenac sodium daily, and G-IV received both 500 IU/kg of vitamin D and 3 mg/kg of diclofenac sodium daily for 14 days. Specimens from rats' stomach were processed for light microscopy. Immunohistochemical examination was carried out to detect inducible nitric oxide synthase (iNOS), proliferating cell nuclear antigen (PCNA), and alpha smooth muscle actin (α-SMA). The morphometric results were analyzed statistically.RESULTS: Gastric sections of G-III displayed inflammatory cellular infiltrations and dilated congested blood vessels. Some of the gastric gland cells showed cytoplasmic vacuolation, dilated gastric pits, and cystic dilatation. There was a significant increased Masson's trichrome stain and a significant decrease in PAS. The mean area percentage of iNOS and α-SMA expression showed a statistically significant increase. The PCNA positive cells were significantly decreased in the isthmus and neck region compared with the control. While in contrast, G-IV prevented the gastric injury by increasing PAS and PCNA but decreasing Masson's trichrome stain, iNOS and α-SMA expression.CONCLUSION: Vitamin D administration prevented the structural alterations of the gastric rat induced by diclofenac sodium.KEYWORDS: diclofenac sodium, α-SMA, iNOS, PCNA, vitamin D

Trolox is more successful than allopurinol to reduce degenerative effects of testicular ischemia/reperfusion injury in rats

Reperfusion surgery following testicular ischemia is a reproductive health threatening status and may result with organ dysfunction in men. The high level of reactive oxygen species (ROS) and cease of blood flow to the testis are the most important reasons of this testicular injury. Until today, numerous experimental studies reported that antioxidants might be efficient to alleviate oxidative stress induced organ dysfunction. For this purpose, in this study, we have investigated the protective effects of xanthine oxidase (XO) inhibitor, allopurinol, and ROS scavenger, trolox, in a comparative perspective in testicular ischemia reperfusion injury subjected rats. Twenty-eight adult male Sprague Dawley rats were divided into four groups of seven animals in each; control, ischemia/reperfusion (I/R), allopurinol and trolox. The rats in control group did not receive any application. Animals in I/R, allopurinol and trolox groups were subjected to 2h testicular reperfusion injury following 5h ischemia. Intraperitoneally (i.p.) 1ml isotonic, 200mg/kg allopurinol and 50mg/kg trolox were administered to the animals in these groups 30min prior reperfusion. At the end of experiment, all animals were sacrificed and blood serum malondialdehyde (MDA) levels were measured. Histological sections were obtained from the testis and stained with hematoxylin and eosin (H&E), proliferating cell nuclear antigen (PCNA) and cleaved caspase-3. Apoptotic index was evaluated with TUNEL Assay. Severe morphological degenerations, increased serum MDA, cleaved caspase-3 and TUNEL Assay positivity rate, but reduced PCNA positivity rate was observed in ischemia and reperfusion group. Morphological degenerations, MDA level, apoptotic index and PCNA positive cell rate were slightly alleviated in allopurinol administered animals compared with ischemia and reperfusion group. Protection with trolox was more successful and the results of the analysis were similar to the control group. Ischemia that leading to testicular torsion is a reproductive health affecting problem and current surgical treatment methods might be insufficient to recover testis. Various types of ROS generating mechanisms in cell are limiting protective potency of allopurinol, and cocktail administration of different ROS inhibitors might be more effective. However, our results indicate that free radical scavenger trolox might be a candidate drug to alleviate degenerative effects of testicular ischemia reperfusion injury. This is the first study that demonstrates antioxidant trolox was more successful than XO inhibitor allopurinol to protect testis against ischemia and reperfusion injury in rats.

THE TOPICAL APPLICATION EFFECT OF NANO-HYDROXYAPATITE VERSUS PLATELET RICH FIBRIN ON THE EARLY STAGES OF EXTRACTION SOCKET HEALING IN RABBITS: HISTOMORPHOMETRIC, HISTOCHEMICAL AND IMMUNOHISTOCHEMICAL STUDY

Introduction: The proper healing of extraction socket is essential for ridge preservation and better sequential prosthetic procedures. Several techniques and materials are developed for acceleration of socket healing. Two of the promising materials for this purpose are nanohydroxyapatite (NHA) and platelet rich fibrin (PRF); their mechanism of action and optimum application techniques are still not fully elucidated. Purpose: To compare between NHA and PRF topical application in the enhancement of early stages of extraction socket healing in rabbits. Materials & Methods: The mandibular first molars were extracted in twenty-four rabbits; the extraction sockets were divided into 3 groups: control group didn’t receive topical treatment. N and P groups received topical application of NHA and PRF respectively. Each group was subdivided into 2 subgroups according to the date of scarification after extraction: 1 week and 2 weeks. The extraction sockets were investigated by hematoxylin and eosin (H&E), Masson trichrome (MT) stains and proliferation cell nuclear antigen (PCNA) marker. The results were illustrated histologically and histomorphometrically. Results: Both NHA and PRF treated groups in both 1 week and 2 weeks periods showed enhanced socket healing, more regular and mature bone in both H&E and MT stains as well as increased PCNA positive cells percentage in comparison to control group. The bone area in both periods and the osteocyte count after 1 week were highest in PRF group followed by NHA group while control group revealed the least values. These differences were statistically non-significant except the percentage of immuno-positive cells. Conclusion: Both NHA and PRF improve the extraction socket healing process with slight preference of PRF particularly in the cellular proliferation in the first week after extraction.

Can lycopene eliminate the harmful effects of hyperoxia in an immature brain?

In addition to protecting cells against free radical harm thanks to its anti-oxidant nature, lycopene strengthens the bonds among cells and improves cell metabolism. This study focuses on analyzing therapeutic effects of lycopene in hyperoxia-induced neurodegenerative disorders in newborn rats. Term newborn rats were divided into four groups as the normoxia control group (group-1), normoxia+lycopene group (group-2), hyperoxia control group (group-3) and hyperoxia+lycopene group (group-4). Group-1 and group-2 were monitored in room air while the group-3 and group-4 were monitored at > 85% O2. The group-2 and group-4 were injected with lycopene intrapertioneally (i.p. ) at 50mg/kg/day while the other groups were injected with corn oil i.p. at the same volume. The rats we sacrificed on the 11th day following the 10-day hyperoxia. The brains were removed and oxidant system parameters were assessed. Injury resulting from hyperoxia was detected in the white matter, cortical regions, and thalamus of the brains. It was observed that the number of apoptotic cells increased and the number of proliferating cell nuclear antigen (PCNA) positive cells decreased in the groups-3 and 4 compared to the group-1. No significant improvement in the number of apoptotic cells and PCNA positive cells was observed in the groups-3 and 4, and apoptosis increased as well. This study found that lycopene, did not show any therapeutic effects for brain damage treatment in newborn rats. In addition, this study demonstrated that lycopene might lead to toxic effects.

Micro-osteoperforations accelerate orthodontic tooth movement by stimulating periodontal ligament cell cycles.

The aim of this study was to investigate the mechanism of how micro-osteoperforations (MOPs) accelerate tooth movement. We focused on inflammation, cell proliferation, and apoptosis ofperiodontal ligament cells and performed immunostaining of MOPs exposed to tumor necrosis factor-alpha (TNF-α), proliferating cell nuclear antigen (PCNA), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) during experimental tooth movement. Eleven-week-old male Wistar rats were divided into 2 groups: (1) 10g of orthodontic force applied to the maxillary first molar (TM) and (2) force application plus 3 small perforations of the cortical plate (TM+MOPs). On days 1, 4, 7, 10, and 14 after force application, we investigated tooth movement and alveolar bone microstructure using microcomputed tomography (n=5). We also determined the expression of TNF-α and PCNA in the pressure sides of periodontal ligaments via an immunohistochemical analysis. The expression of apoptotic cells was also determined by the TUNEL method. The tooth movement in the TM+MOPs group was significantly greater on days 4 to 14 than in the TM group. The TM+MOPs group showed statistically significant decreases in bone volume/tissue volume ratio and bone mineral density compared with the TM group. The ratios of TNF-α positive cells in the TM+MOPs group were increased on days 1, 4. 7, and 10 compared with the TM group. The ratios of PCNA positive cells in the TM+MOPs group were increased on days 1, 4, and 7 compared with the TM group, and the ratios of TUNEL positive cells in the TM+MOPs group were increased on days 1 and 7 compared with the TM group. These results suggest that MOPs may accelerate tooth movement through activation of cell proliferation and apoptosis of periodontal ligament cells.

Effect of lentivirus-mediated small interfering RNA on mitogen- and stress-activated protein kinase 1 in spinal cord injury of rats

To investigate the expression changes and the repair effect of mitogen and stress- activated protein kinase 1 (MSK1) on spinal cord injury (SCI) in rats. One hundred and twenty male Sprague Dawley (SD) rats (weighing 220-250 g) were used for the study, 70 of them were randomly divided into sham-operation group and SCI group ( n=35), the rats in SCI group were given SCI according to Allen's method, and the sham-operation group only opened the lamina without injuring the spinal cord; spinal cord tissue was collected at 8 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, and 7 days after invasive treatment, each group of 5 rats was used to detect the expression of MSK1 and proliferating cell nuclear antigen (PCNA) by Western blot assay. Another 20 SD rats were grouped by the same method as above ( n=10). In these rats, a negative control lentiviral LV3NC dilution was injected at a depth of approximately 0.8 mm at the spinal cord T 10 level. The results of transfection at 1, 3, 5, 7, and 14 days after injection were observed under an inverted fluorescence microscope to determine the optimal transfection time of the virus. The other 30 SD rats were randomly divided into group A with only SCI, group B with a negative control lentiviral LV3NC injected after SCI, and group C with MSK1 small interfering RNA (siRNA) lentivirus injected after SCI, with 10 rats each group. The Basso, Beatlie, Bresnahan (BBB) score of hind limbs was measured at 1, 3, 5, 7, and 14 days after treatment; spinal cord tissue collected at the optimal time point for lentivirus transfection was detected the expression changes of MSK1 and PCNA by Western blot and the localization by immunofluorescence staining of MSK1 and PCNA proteins. Western blot assay showed that there was no significant changes in the expression of MSK1 and PCNA at each time points in the sham-operation group. In the SCI group, the expression of MSK1 protein was gradually decreased from 8 hours after injury to the lowest level at 3 days after injury, and then gradually increased; the expression change of PCNA protein was opposite to MSK1. The expression of MSK1 in SCI group was significantly lower than that in the sham-operation group at 1, 2, 3, and 5 days after injury ( P<0.05), and the expression of PCNA protein of SCI group was significantly higher than that of the sham-operation group at 8 hours and 1, 2, 3, 5, and 7 days after injury ( P<0.05). The fluorescence expression of both the SCI group and the sham-operation group has be found and peaked at 7 days. There was a positive correlation between fluorescence intensity and time in 7 days after transfection. With the prolongation of postoperative time, the BBB scores of groups A, B, and C showed a gradually increasing trend. The BBB score of group C was significantly lower than those of groups A and B at 5, 7, and 14 days after treatment ( P<0.05). After transfection for 7 days, Western blot results showed that the relative expression of MSK1 protein in group C was significantly lower than that in groups A and B ( P<0.05); and the relative expression of PCNA protein was significantly higher than that in groups A and B ( P<0.05). Immunofluorescence staining showed that MSK1 was expressed in the nuclei of the spinal cord and colocalized with green fluorescent protein, neuronal nuclei, and glial fibrillary acidic protein (GFAP). The relative expression area of MSK1 positive cells in group C was significantly higher than that in group B ( P<0.05), and the relative expression areas of PCNA and GFAP positive cells were significantly lower than those in group B ( P<0.05). Lentivirus-mediated MSK1 siRNA can effectively silence the expression of MSK1 in rat spinal cord tissue. MSK1 may play a critical role in the repair of SCI in rats by regulating the proliferation of glial cells.

Immunohistochemical Study of the Ameliorative Effect of Vitamin E on Liver Regeneration after Different Periods of Partial Hepatectomy

The liver is almost unique in its capacity for regeneration after hepatectomy but the exact mechanisms are not yet fully clarified. Antioxidants have been shown to promote liver regeneration after major hepatectomy. The present study evaluated the ameliorative effect of vitamin E administration on the liver regeneration after different periods of partial hepatectomy (PH) in rats. Fifty-six adult male albino rats were divided into three groups: Control sham operated group; partially hepatectomized group which were divided into three subgroups sacrificed at 1day, 3 days and 7days after the operation respectively; Partially Hepatectomized group with vitamin E pretreatment before PH where the rats were given a daily oral dose of vitamin E until the time of sacrifice of the rats. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and labeling index were demonstrated. After PH, the PCNA positive hepatocytes and the PCNA labeling indices were significantly high after the 1st day and then much decreased after the 3rd day, to be followed by a slight increase at the 7th day. Vitamin E pretreatment in PH rats resulted in a decrease in PCNA positive cells and its labeling indices in the 1st day with a gradual increase in the 3rd and 7th days. Vitamin E has an inhibitory effect in the first 24 hours on liver regeneration followed by stimulatory effect at the third and seventh days after PH. These data indicated that vitamin E pretreatment has an important role in regulation and enhancement of liver regeneration after PH.

Effects of denatured collagen type Ⅰ on differentiation of human fibroblasts into myofibroblasts

Objective: To investigate the effects of denatured collagen type Ⅰ on differentiation of human fibroblasts into myofibroblasts. Methods: A small amount of normal skin donated by burn patients undergoing scar surgery was collected. Human fibroblasts were obtained by method of explant culture and then sub-cultured. The fourth passage of cells were used in the following experiments. (1) Fibroblasts were divided into normal collagen group and denatured collagen group according to the random number table, with 10 wells in each group. Fibroblasts in normal collagen group were cultured on normal collagen type Ⅰ coated coverslips. Fibroblasts in denatured collagen group were cultured on denatured type Ⅰ collagen coated coverslips. Expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemical method, and the percentage of PCNA positive cells was calculated. (2) Another batch of fibroblasts were grouped and treated as in (1), with 12 wells in each group. Proliferation activity of cells was determined with methyl-thiazolyl-tetrazolium colorimetry method. (3) Another batch of fibroblasts were grouped and treated as in (1), and the microfilament morphology of cells was observed by rhodamine-phalloidin staining. (4) Another batch of fibroblasts were grouped and treated as in (1). Expression of α smooth muscle actin (α-SMA) of cells was detected by immunohistochemical method, and expression of OB-cadherin of cells was detected by immunofluorescence method. (5) Another batch of fibroblasts were divided into normal collagen, denatured collagen, and common coverslips groups according to the random number table, with 6 wells in each group. Fibroblasts in normal collagen and denatured collagen groups were treated as in (1), while fibroblasts in common coverslips group were cultured on coverslips without collagen coating. Expressions of α-SMA and OB-cadherin of cells were determined with Western blotting. (6) Another batch of fibroblasts were grouped and treated as in (5), and then the mRNA expressions of collagen type Ⅰ, collagen type Ⅲ, and α-SMA of cells were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with t test, one way analysis of variance, and least-significant difference test. Results: (1) The percentage of PCNA positive cells in denatured collagen group was (83±9)%, significantly higher than (29±9)% in normal collagen group (t=13.53, P<0.01). (2) The proliferation activity of fibroblasts in denatured collagen group was 0.32±0.06, significantly higher than 0.25±0.05 in normal collagen group (t=3.06, P<0.01). (3) The microfilament of fibroblasts in normal collagen group was arranged vertically and in parallel way, paralleling the long axis of cells. The microfilament of fibroblasts in denatured collagen group was denser and thicker. (4) Most fibroblasts in normal collagen group showed long shuttle-like shape typically. Morphology of fibroblasts in denatured collagen group changed, and cells were obviously spreading. Expressions of α-SMA and OB-cadherin of fibroblasts in denatured collagen group were stronger than those in normal collagen group. (5) Expressions of α-SMA of fibroblasts in denatured collagen, normal collagen, and common coverslips groups were respectively 1.69±0.41, 0.89±0.27, and 1.46±0.42. Expression of α-SMA of fibroblasts in denatured collagen group was significantly higher than that in normal collagen group (P<0.01). Expressions of OB-cadherin of fibroblasts in denatured collagen, normal collagen, and common coverslips groups were respectively 5.17±0.28, 2.21±0.10, and 4.01±0.56. Expression of OB-cadherin of fibroblasts in denatured group was significantly higher than that in normal collagen group (P<0.01). (6) There was no significant difference in mRNA expression of collagen type Ⅰ of fibroblasts in denatured collagen, normal collagen, and common coverslips groups (F=2.71, P>0.05). The mRNA expressions of collagen type Ⅲ and α-SMA of fibroblasts in normal collagen group were significantly lower than those in denatured collagen group (P<0.01). Conclusions: Denatured collagen type Ⅰ may influence the activity of fibroblasts, thus inducing fibroblasts differentiating into myofibroblasts.

Histological Alterations from Condyle Repositioning with Functional Appliances in Rats.

This study was designed to assess the morphological and histological alterations of the condyle of rats undergoing forward mandibular repositioning via functional appliance. Functional appliances were mounted onto the upper jaws of rats. Morphological analysis was conducted on micro-CT images of sacrificed animals. Histological changes in condyle were examined by immunohistochemistry using proliferating cell nuclear antigen (PCNA), matrix metalloproteases (MMPs), vascular endothelial growth factor (VEGF), tissue inhibitors of matrix metalloproteinases (TIMP-1), interleukin 1b (IL-1β), Aggrecan and Type II collagen. Osteoclast activity was identified by tartrate-resistant acid phosphatase (TRAP) staining. Morphological analysis confirmed the forward positioning of the condyles of rats by the appliance, but the position gradually returned to normal on days 14 after treatment. An increase in PCNA positive cells was observed in the posterior region of the condyles on days 7, whereas PCNA positive cells decreased in the anterior region. Aggrecan and Type II collagen localization increased in the posterior region throughout the entire period, but decreased in the anterior region on days 14. In both regions, IL-1β and VEGF localization was significantly increased for 14 days while MMPs localization was evident throughout the entire period. The TRAP positive cells were significantly elevated on days 3 and 7. These results suggest that the functional appliance therapy induces significant morphological and histological changes in the anterior and posterior regions of the condyle and subsequently causes adaptive cellular functions such as chondrocyte differentiation and cartilage matrix formation.

GIT1 gene deletion delays chondrocyte differentiation and healing of tibial plateau fracture through suppressing proliferation and apoptosis of chondrocyte

BackgroundAlthough tibial plateau fracture is an uncommon injury, its regulation is challenging and there are some influencing factors, including the effects of severe bone displacement, depression and cancellous bone cartilage, and inevitable cartilage damage. And GIT1 plays an important role in bone mass and 78 osteoblast cell migration.MethodsThe study used 72 C57/BL6 mice. A tibial plateau fracture model was established by using mice with the same number of GIT1 gene deletions (the experimental group) and their wild-type littermates (the control group). Joint and bone callus recovery were evaluated by X-ray and CT thin layer scans. Micro CT assay and histomorphometry were conducted in order to evaluate the volume of newly formed blood vessels. Type II collagen expression in tibial tissues after tibial plateau fracture were detected by immunohistochemistry after 7, 14 and 21 days. The number of proliferating cell nuclear antigen (PCNA) positive cells after tibial plateau fracture was tested by immunohistochemistry after 14 and 21 days. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was conducted after 14 and 21 days in order to test chondrocyte apoptosis in tibial tissues after tibial plateau fracture.ResultsThe GIT1 gene deletion group mice spent less time on the rotating rod than the control group mice (P < 0.05). Compared with the control group, postoperative recovery was retarded, because GIT1 gene deletion slowed down neovascularization after tibial plateau fracture (P < 0.05). Compared with the control group, mouse type II collagen expression significantly decreased in the GIT1 gene deletion group, and the proportion of PCNA positive cells significantly decreased (P < 0.05). The TUNEL results indicate that GIT1 gene deletion led to reduced chondrocyte apoptosis.ConclusionGIT1 gene deletion can inhibit chondrocyte proliferation and apoptosis during the recovery of tibial plateau fracture, so as to delay chondrocyte differentiation and tibial plateau fracture healing.

Local transplantation of human thrombomodulin gene-modified endothelial progenitor cells to prevent restenosis after angioplasty

Objective To investigate whether restenosis can be inhibited by human thrombomodulin (hTM) gene-modified endothelial progenitor cells (EPCs) after angioplasty. Methods EPCs derived from rabbit periphery blood were modified with hTM gene using lentiviral vector and were incubated with Fe2O3-arginine. Thirty-six New Zealand rabbits were randomly divided into 3 groups. Angioplastywas underwent by Fogarty ballooncatheter via catheterization of right common carotid artery, and then hTM-EPCs, EPCs or equal volume of normal saline were transplanted to the injury sites with blood flow blocked respectively. Histology and Western blotting were performed on 1th week for 4 rabbits from each group to evaluate cell adherence. Magnetic resonance (MR) was performed on 4th and 8th week after transplantation to evaluate restenosis. All of the rest rabbits were sacrificed at the end of 8th week after injury and the injured vessels were harvested for hematoxylin and eosin (HE), elastic tissue and proliferating cell nuclear antigen (PCNA) staining to observe the vascular wall structure and degree of intimal hyperplasia and smooth muscle proliferation. Results Prussian blue staining showed iron-labeled cells distributed in the injured artery intima. Western blotting indicated that injured vascular transplanted with hTM-EPCs overexpressed thrombomodulin than other two groups. MR showed that the stenotic degree of the lumen in hTM-EPCs group was less serious than other two groups. The histopathological study on 8th week showed that the neointimal/media ratio (N/M) in hTM-EPCs group, EPCs group and control group was 0.348±0.040, 0.704±0.060 and 1.294±0.051, respectively (P=0.000); the ratio of restenosis in each group was (11.4±2.3)%, (24.3±4.8)% and (35.2±9.4)%, respectively (P=0.005); and the PCNA positive cell index in each group was (12.3±3.9)%, (23.4±4.2)% and (49.3±6.8)%, respectively (P=0.000). Conclusion The local transplantation of hTM-EPCs can prevent restenosis through Fogarty balloon after angioplasty in rabbit model. Key words: Thrombomodulin; Endothelial progenitor cells; Balloon injury; Restenosis

Gastroprotective effects of montelukast and Nigella sativa oil against corticosteroid-induced gastric damage: they are much more than antiasthmatic drugs.

Corticosteroids are used to treat a variety of diseases like bronchial asthma. However, long-term corticosteroids have a gastric ulcerogenic potential. Montelukast (MTK) and Nigella sativa oil (NSO) are used in treatment of bronchial asthma. Previous studies showed that MTK and NSO had gastroprotective effects in other models of gastric ulcer. The present study assesses synergistic gastroprotective effects of both drugs in dexamethasone (DXM)-induced gastric damage. Fifty male rats were divided into 5 groups: normal control (I), DXM group (II), MTK + DXM group (III), NSO + DXM group (IV), MTK + NSO + DXM group (V). After 7 days, stomachs were removed for biochemical analysis and histological examinations. Significant increases in malondialdehyde (MDA) level, superoxide dismutase (SOD) activity, myeloperoxidase (MPO) activity, and proliferating cell nuclear antigen (PCNA) positive cells, with significant decreases in mucus secretion were detected in DXM-treated group compared with group I. Meanwhile, significant decreases of MDA level, MPO activity, and PCNA positive cells and significant increases in mucus secretion were detected in treated groups compared with group II. SOD activity significantly decreased in group V compared with group II. We could conclude that administration of either MTK or NSO or both with DXM counteracts DXM-induced gastric lesions.

Tacrolimus-Eluting Suture Inhibits Neointimal Hyperplasia: An Experimental In Vivo Study in Rats

Neointimal hyperplasia (NIH) remains one of the leading causes of graft failure after vascular anastomoses. Cytotoxic drugs, such as rapamycin and tacrolimus, have been shown to inhibit the development of NIH. In this study, the aim was to test the impact of a sustained releasing tacrolimus-chitosan-eluting suture on the development of NIH in a rat model. After tacrolimus-chitosan coating of a 7/0 polyvinylidene difluoride (PVDF) Trofilen® suture, the tacrolimus concentration on the coated suture and invitro release trials were performed spectrophotometrically. Twelve Wistar rats were included. After midline laparotomy, a 7-8mm longitudinal aortotomy in the infrarenal aorta was made and then closed by a bare 7/0 PVDF (group C, n=6) and a 7/0 tacrolimus-chitosan coated PVDF suture (0.65μg/cm tacrolimus [0.9wt%]+1.82μg/cm chitosan [2.28wt%]) (group T, n=6). After 1 month, rats were sacrificed and aortotomy sites were examined histologically by ratio of intimal area (including neointima) and immunohistochemically by α-smooth muscle actin (ASMA) and proliferating cell nuclear antigen (PCNA) immunostaining. The PCNA positive cells were indexed to total cell number and expressed as percentage. Invitro tacrolimus release tests for a 7/0 tacrolimus-chitosan coated PVDF suture were confirmed for 1month without an initial burst release. Endothelialisation over the aortotomy line occurred in both groups. The area of neointima was significantly reduced in group T compared with group C (ratio 0.22±0.12 vs. 0.42±0.11; p=.017) 1 month post-operatively. Likewise, the percentage of PCNA immunostaining significantly decreased in group C compared with group T (3.83±2.85% vs. 11.17±7.78%; p=.026). The cells constituting NIH were positive for ASMA immunostaining. Tacrolimus-chitosan-eluting suture is shown to be an effective way to reduce NIH without interfering with normal endothelialisation.

Protective effect of vitamin D against hyperoxia-induced lung injury in newborn rats.

Preterm infants have risks of developing vitamin D deficiency. Thus we aimed to investigate the effect of vitamin D on hyperoxia-induced lung injury in newborn rats. Full term rat pups were included in the study 12-24 hr after delivery. The pups were randomly divided into eight groups as follows: normoxia control group (NC), normoxia plus vitamin D group (ND1, 1 ng/gr/day vitamin D), normoxia plus vitamin D group (ND2, 3 ng/gr/day vitamin D), normoxia plus vitamin D group (ND3, 5 ng/gr/day vitamin D), hyperoxia control group (HC), hyperoxia plus vitamin D group (HD1, 1 ng/gr/day vitamin D), hyperoxia plus Vitamin D group (HD2, 3 ng/gr/day vitamin D), hyperoxia plus vitamin D group (HD3, 5 ng/gr/day vitamin D). The histopathological effects of vitamin D were assessed by alveolar surface area (with mean linear intercept (MLI) method), apoptosis index and proliferating cell nuclear antigen (PCNA) index. MLI values were significantly lower among three groups (HD1: 83.93 ± 1.95 μm, HD2: 81.76 ± 1.68 μm, and HD3: 82.33 ± 1.87 μm) when compared with HC group (92.98 ± 2.09 μm) (P = 0.001, P = 0.0004, P = 0.002, respectively). Apoptotic cell index were significantly lower among three treatment groups (HD1: 1.455 ± 0.153, HD2: 0.575 ± 0.079, and HD3: 0.700 ± 0.105) when compared with HC group (2.500 ± 0.263) (P = 0.001, P = 0.001, P = 0.001, respectively). Although PCNA positive cell index did not change in HD1 group (0.132 ± 0.008) (P > 0.05), there were significant increases in HD2 (0.277 ± 0.026) and HD3 (0.266 ± 0.018) group when compared with HC group (0.142 ± 0.010) (HD2 P = 0.001, HD3 P = 0.001). Vitamin D seems to protect hyperoxia-induced lung injury in newborn rats. Pediatr Pulmonol. 2017;52:69-76. © 2016 Wiley Periodicals, Inc.

Preliminary study of artificial synthetic E-selectin inhibits the proliferation mechanism of vascular smooth muscle cells in rats

Objective To preliminary study the mechanism of artificial synthetic E-selectin inhibiting the proliferation of vascular smooth muscle cells in rats. Methods Ninety adult male SD rats were randomly divided into 3 groups: a control group, an operation group, and a treatment group (n=30 in each group). The right common carotid artery was made a longitudinal incision and interrupted suture in the operation group and the treatment group, and the control group did not make vascular incision and suture. Artificial synthetic E-selectin and the same amount of saline were injected into the femoral veins of the rats respectively in the treatment group and the operation group after vascular suture. On day 1, 3, 7, and 14 after procedure, ELISA was used to detect the concentrations of serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The vascular tissue at the suture was taken. Western blot and RT-PCR were used to detect the protein level of NF-κB p65 and mRNA expression. Electrophoretic mobility shift assay was used to detect the NF-κB transcriptional activity. Immunohistochemical staining was used to detect the positive cell rate of proliferating cell nuclear antigen (PCNA). Results The concentrations of serum TNF-α and IL-6 and the levels of NF-κB p65 protein and mRNA began to increase at day 1 after vascular suture in the operation group and the treatment group. They reached the peak at day 3, and they were still higher than those in the control group at day 7 (P 0.05). The NF-κB transcription activity in the operation group was higher than that in the treatment group at day 3 after suture (P<0.01). The PCNA positive cells began to express at day 3 after vascular suture in the operation group and the treatment group. The expression of PCNA positive cells in the control group was not obvious or no expression. There were significant differences among the 3 groups (P<0.05). They still expressed high at day 7 and 14 (P<0.01), while the expression of PCNA positive cells was lower than that in the treatment group over the same period (P<0.01). Conclusion Artificial synthetic E-selectin can inhibit the proliferation of vascular smooth muscle cells by inhibiting NF-κB activity. Key words: E-selectin; NF-kappa B; Myocytes, smooth muscle; Cell proliferation; Rats; Vessels

Steamed root of Rehmannia glutinosa Libosch (Plantaginaceae) alleviates methotrexate-induced intestinal mucositis in rats

Ethnopharmacological relevanceIntestinal mucositis induced by chemotherapy is a severe clinical problem in cancer patients that currently lack effective interventions. In traditional Chinese medicine, chemotherapeutic toxicity is diagnosed as Qi and Yin deficiency, and steamed rehmannia root (SRR) is frequently prescribed to these patients. Whether SRR can prevent the adverse effects remains to be confirmed experimentally. The present study used a rat model to investigate potential efficacy and action mechanisms of SRR in attenuating the adverse effects caused by chemotherapy. Materials and methodsIntraperitoneal injection of a single dose of anti-metabolite methotrexate (MTX, 25mg/kg) was given to adult Wistar rats, which also received oral gavage of water or SRR (1.08g/kg twice daily 3 days before and 4 days after MTX treatment), or calcium folinate (CF, a clinically used MTX antidote as a comparison, at 1mg/kg twice daily 36h after MTX treatment), or SRR and CF in combination. Animals were sacrificed 4 days after MTX treatment. Complete blood cell counting was carried out. Jejunum was analyzed histologically for mucosal damage, immunohistochemically for proliferating cell nuclear antigen (PCNA), and biochemically for thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH), as well as for tumor necrosis factor alpha (TNF-α). ResultsMTX treatment led to weight loss, leucopenia, polycythemia, increase in large thrombocyte ratio, intestinal villus atrophy, crypt loss and reduction in PCNA positive crypt cells, increases in mucosal TBARS and TNF-α and decrease in GSH. All these alterations were inhibited by SRR administration except leucopenia, and the effects of CF or CF plus SRR supplementation were found to be inferior to those of SRR. ConclusionsSRR can alleviate MTX-induced gut mucositis, which may be achieved by inhibiting MTX-induced oxidative stress and inflammatory response. These findings support the application of SRR in chemotherapy but not the combined application of SRR and CF.