Number Of Ki67-positive Cells Research Articles

A Rat Model for Oral Mucositis Induced by a Single Administration of 5-Fluorouracil.

This study aimed to develop a reliable chemotherapy-induced oral mucositis (CIOM) rat model by intraperitoneally administering a single dosage of 5-fluorouracil (5-FU) combined with a chemical stimulus. The 5-FU dosage for CIOM development was determined by the survival rate of rats administrated 160 mg/kg, 200 mg/kg, and 240 mg/kg of 5-FU. Thirty rats were assigned to normal control (NC) and three experimental groups: i) ulcer formation without 5-FU administration (PBS/U+), ii) 5-FU administration without ulcer formation (5-FU/U-), and iii) ulcer formation after 5-FU administration (5-FU/U+). White blood cell count and weight were measured at the day of 5-FU administration (D0), ulcer formation (D2), and two days after ulcer formation (D4). The oral mucosa for histologic evaluations was obtained two (D4) and five days (D7) after ulcer formation. The 5-FU dosage for CIOM development was 200 mg/kg. White blood cell count (WBC) counts and weight of rats were significantly lower in 5-FU/U- (WBC, p<0.001; weight, p=0.002) and 5-FU/U+ (WBC, p<0.001; weight, p<0.001) groups compared to those in the NC group at D4. The number of Ki-67 positive cells in the oral epithelium was lower in 5-FU/U+ group compared to that in NC (p<0.001) and PBS/U+ (p=0.047) groups at D7. Single administration of 200 mg/kg of 5-FU combined with a chemical stimulus can lead to an immune-suppressive status, failure of weight gain, and impairment of epithelium regeneration as observed in a CIOM rat model.

Salvianolic acid B exerts an anti-hepatocellular carcinoma effect by regulating the Hippo/YAP pathway and promoting pSmad3L to pSmad3C simultaneously

Salvianolic acid B (Sal B) is a component obtained from Salvia miltiorrhiza and is empirically used for liver diseases. The TGF-β/Smad and Hippo/YAP pathways may interact with each other in hepatocellular carcinoma (HCC). Previously, we found that Sal B mediates the TGF-β/Smad pathway in mice and delays liver fibrosis-carcinoma progression by promoting the conversion of pSmad3L to pSmad3C, but the effect of Sal B on the Hippo/YAP pathway has not been determined. Therefore, we used a DEN/CCl4/C2H5OH-induced liver cancer model in mice to analyze liver index and tumor incidence, detect AST and ALT serological markers, observe liver pathology and the number of Ki67-positive cells to evaluate the anti-HCC effect of Sal B in vivo. We used a TGF-β1-induced HepG2 cell model, and applied an MST1/2 inhibitor, XMU-MP-1, to detect the changes in pSmad3C/pSmad3L signaling induced by MST1/2 inhibition. Sal B significantly inhibited tumorigenesis in DEN/CCl4/C2H5OH-induced mice in vivo, and suppressed the growth of HepG2 cells by inhibiting cell proliferation and migration in vitro. Here, our study also validated the role of Sal B in reversing XMU-MP-1-induced proliferation and migration of HepG2 cells in vitro. Most importantly, we elucidated for the first time the potential mechanism of Sal B against HCC via the Hippo/YAP pathway, which may be specifically related to upregulation of MST1 and inhibition of its downstream effector protein YAP. In conclusion, these findings indicate that Sal B possesses anti- HCC effects both in vivo and in vitro by regulating the Hippo/YAP pathway and promoting pSmad3L to pSmad3C synchronously.

Magnolol Suppresses Breast Cancer Cells via Regulating miR-140-5p/TLR4 Signaling Pathway

Objective: To discuss Mag effects and relative mechanisms in breast cancer treatment by vitro study. Materials and methods: In first step, using difference concentrations of Mag to treat breast cancer cell lines; In next step, the cell liens were divided into NC, Mag and Mag+si-miRNA group. Using MTT to measure cell proliferation rates; using TUNEL and flow cytometry to evaluate apoptosis cell number and rate; measuring invasion cell number and wound healing rate using transwell or wound healing; evaluating relative gene expressions using RT-qPCR and WB assay. Results: Cell proliferation rates, invasion cell number, Ki67 positive cell number, wound healing rates significant depressed (P &lt; 0.05) and cell apoptosis rate and apoptosis cell number significantly increased (P &lt;0.05, respectively), meanwhile, miR-140-5p, TLR4, MyD88 and NF-κB(p65)gene significantly changed (P &lt; 0.05) and TLR4, MyD88 and NF-κB(p65) protein significant down-regulation (P &lt; 0.05). However, with si-miRNA which inhibited miR-140-5p supplement, the cell biological activities significantly increased (P &lt;0.001), with miR-140-5p significant down-regulation, TLR4, MyD88 and NF-κB(p65) significantly up-regulation (P &lt; 0.001). Conclusion: Mag had anti-tumor effects to breast cancer via miR-140-5p/TLR4 axis by vitro cell experiment.

Novel ROCK Inhibitors, Sovesudil and PHP-0961, Enhance Proliferation, Adhesion and Migration of Corneal Endothelial Cells

The loss or dysfunction of human corneal endothelial cells (hCEnCs) is a leading cause of blindness due to corneal failure. Corneal transplantation with a healthy donor cornea has been the only available treatment for corneal endothelial disease. However, the need for way to regenerate the CEnCs has been increased due to the global shortage of donor corneas. The aim of the study is to investigate whether novel Rho-kinase (ROCK) inhibitors can induce the cultivation and regeneration of hCEnCs. Cultured hCEnCs were treated with Y-27632, sovesudil, or PHP-0961 for 24 h. Cellular responses, including cell viability, cytotoxicity, proliferation, and Ki67 expression with ROCK inhibitors were evaluated. We also evaluated wound healing and cell adhesion assays. Porcine corneas were used ex vivo to evaluate the effects of Y-27632, sovesudil, and PHP-0961 on wound healing and regeneration. We performed live/dead cell assays and immunofluorescence staining for SRY (sex determining region Y)-box 2 (SOX2), β-catenin, and ZO-1 on porcine corneas after ROCK inhibitor treatments. Cell viability, cell proliferation rate, and the number of Ki67-positive cells were higher in Y-27632, sovesudil and PHP-0961 treated cells compared to the control. There was no difference in LDH cytotoxicity test between any groups. Cells treated with Y-27632, sovesudil and PHP-0961 showed faster migration, wound healing, and cell adhesion. In the porcine ex vivo experiments, wound healing, the number of live cells, and SOX2-positive cells were higher in Y-27632, sovesudil and PHP-0961 treated corneas. In all experiments, sovesudil and PHP-0961, the novel ROCK inhibitors, were equal or superior to the results of the ROCK inhibitor positive control, Y-27632. In conclusion, sovesudil and PHP-0961, novel ROCK inhibitors have the capacity to regenerate hCEnCs by enhancing cell proliferation and adhesion between cells.

Effects of exosomes from hepatocyte growth factor-modified human adipose mesenchymal stem cells on full-thickness skin defect in diabetic mice

Effects of exosomes from hepatocyte growth factor-modified human adipose mesenchymal stem cells on full-thickness skin defect in diabetic mice

Abstract 15359: Multi-Omics Integration Reveals Mechanosensitive Regulatory Pathways in Cardiac Fibroblasts

Introduction: Regionalized myocardial fibrosis exists in the inferobasal myocardium of patients undergoing surgical repair for Mitral Valve Prolapse, and supports a process in which cells within mechanically stressed myocardial regions alter their phenotype in order to increase tissue stiffness, eventually causing establishment of a fibrotic scar. How cells behave in different fibromechanical environments is not currently well understood. Hypothesis: Integrating RNA Seq and ATAC Seq datasets will identify mechanosensitive regulatory pathways in cardiac fibroblasts. Methods: Primary human cardiac fibroblasts (HCFs) were cultured on stiff (TC Plastic, ~2GPa), as well as soft (2kPa) substrates coated with type 1 collagen. Following 48 hours in serum-free conditions, HCFs were lysed and subjected to immunofluorescence (IF), Western blotting (WB), RNA Seq, and ATAC Seq. Differential analysis of RNA Seq, and ATAC Seq data was performed, followed by an integration analysis which linked differentially expressed genes to chromatin that opened/closed within 10 kB. Finally, motif enrichment analysis was performed using the online bioinformatics package Homer. Results: IF and WB revealed that HCFs cultured on a stiff substrate expressed markers of fibroblast activation (alpha SMA, P&lt;0.0001), as well as increased amounts of Connexin 43 (P&lt;0.0001). Additionally, there were higher numbers of Ki-67 positive cells (7.2% vs 2.1% P=0.001). RNA Seq revealed differential changes in 4324 genes (FDR adjusted P&lt;0.05), several of which were subsequently validated using western blots. ATAC Seq identified 5855 chromatin regions which opened/closed in response to different mechanical environments in fibroblasts (adjusted P&lt;0.05). Integrating RNA Seq and ATAC Seq datasets revealed 803 Chromatin changes that fell within 10kb of differentially expressed genes, with a predicted enrichment of TEAD binding sites in differentially opened chromatin (P=1.0*10-40). Conclusions: The mechanical environment plays a major role in cellular phenotype and directly regulates chromatin organization and expression of pro-fibrotic genes, with a possible role for TEAD as a driver of fibroblast activation.

Adverse effects of maternal retinyl palmitate, a vitamin A compound, on the fetal liver.

Background: Consuming high doses of vitamin A during pregnancy may lead to malformations in the offspring. Some reports state that low doses that do not cause macroscopic abnormalities may result in mental and behavioral disorders. However, there are few studies on the microscopic effects of these doses on the organism. Objective: The aim was to investigate the effects of early prenatal exposure to different doses of oral vitamin A on the fetal liver. Materials and methods: Twenty-five pregnant rats, divided into five groups, received oral vitamin A at doses of 10,000, 50,000, 100,000, and 200,000 IU/kg between days 10 and 12 of gestation. The fetuses were collected on day 19 of gestation, their livers were dissected, and histology, apoptosis, and proliferation were examined by hematoxylin-eosin, TUNEL assay, and Ki67 immunolabeling using stereological methods. Results: Vitamin A decreased fetal liver volume, the number of Ki67-positive cells per unit volume, and the total number of hepatocytes at all doses except 10,000 IU/kg (p<0.001). Consequently, apoptosis was significantly higher in the groups receiving 100,000 and 200,000 IU/kg vitamin A (p<0.001). Conclusion: Our study shows that vitamin A administered during gestation days 10-12 has a suppressive effect on the developing rat liver when the dose exceeds 10,000 IU/kg, probably due to increased apoptosis and suppressed cell division.

An ex-vivo study to investigate the potential of Staphylococcus epidermidis lysate to improve wound healing in diabetic patients

Appropriate treatment of non-healing diabetic foot ulcers is challenging as the whole skin repair process is complicated. Current therapeutic strategies have to be evolved for better efficacy. Staphylococcus epidermidis (S. epidermidis) is a commensal having probiotic properties. The wound healing potential of its lysate was explored using skin biopsies. Full-thickness skin, obtained from individuals with and without diabetes, was wounded using an excisional punch. Skin explants were cultured using a serum-free medium in the presence and absence of S. epidermidis lysate. The ability of the lysate to stimulate re-epithelialisation was assessed in an ex-vivo human skin model of wounding from patients with and without diabetes. Epithelial tongue length was significantly reduced in diabetic wounds in comparison to non-diabetic samples, even after 12 days post-wounding. Re-epithelialisation was augmented considerably in S. epidermidis lysate-treated wounds ex-vivo. In non-diabetic samples, the increase was twofold greater but in diabetic samples, only a slight enhancement in tongue length was observed in comparison to the control (untreated sample). The epidermal thickness also increased 7.7-fold in treated wounds in comparison to 4.4-fold in untreated samples. The cell number per unit area showed a significant increase in comparison to untreated control and the number of Ki-67 positive cells was slighter greater indicating increased expression of the proliferation marker in the newly formed epithelial tongue area in lysate-treated samples (both diabetic and non-diabetic). S. epidermidis lysate promotes re-epithelialisation in both diabetic and non-diabetic skin biopsies and may be used to accelerate healing in chronic diabetic ulcer wounds. Its therapeutic potential needs to be explored further with in vivo and molecular studies.

Low Birth Weight Intensifies Changes in Markers of Hepatocarcinogenesis Induced by Fructose Consumption in Rats.

Intrauterine growth restriction (IUGR) due to fetal exposure to glucocorticoid excess results in metabolic inflexibility and hepatic steatosis upon nutritional stress during adulthood. We previously demonstrated that rats born to dexamethasone (DEX)-treated mothers developed hepatic steatosis when exposed to 10% fructose solution during adult life. Persistent triacylglyceride (TAG) accumulation in the liver, in turn, is a feature of non-alcoholic fatty liver disease (NAFLD), which serves as a risk factor for non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). In the present study, we demonstrate that the combination of IUGR and fructose treatment during adulthood also results in increased hepatic myeloperoxidase (MPO) activity, AKT phosphorylation and serum aspartate transaminase. Growth-restricted rats also presented reduced hepatic TRIB3 and GADD45a after fructose treatment. Other markers of cell proliferation, such as Cyclin D, PCNA, Hgf and Hspa4/Hsp70 expression and the number of Ki-67 positive cells, were all increased in the liver of growth- restricted rats treated with fructose. On the other hand, the combination of IUGR and fructose treatment during adult life reduced the levels of IGF-1. In conclusion, our data indicate that after exposure to fructose, adult rats subjected to dexamethasone-induced IUGR display exacerbated molecular changes in markers of NASH and HCC.

The value of Alcian blue periodic acid Schiff staining and Ki-67 expression in diagnosing gastric reactive epithelial hyperplasia and dysplasia

Objective: To investigate the clinicopathological characteristics of reactive epithelial hyperplasia and dysplasia in the stomach, as well as the clinical value of mucin special staining and proliferating cell nuclear antigen (Ki-67) in distinguishing the two gastric lesions. Methods: The clinical pathological data of 63 patients with gastric reactive epithelial hyperplasia, 54 patients with low-grade dysplasia, and 63 patients with high-grade dysplasia diagnosed from May 2018 to May 2021 in Beijing Chaoyang Hospital, Capital Medical University, Beijing, China were analyzed. Alcian blue periodic acid Schiff (AB-PAS) and Ki-67 staining were performed to examine the mucin staining pattern, number of Ki-67 positive cells, Ki-67 staining patterns in the three groups of lesions, and histopathologic characteristics. Results: The positive rates of AB-PAS in the reactive epithelial hyperplasia and gastric dysplasia groups were 87.3%(55/63) and 10.3%(12/117), respectively. The expression of AB-PAS in the reactive epithelial hyperplasia was gradually increased from the base to the surface of the epithelium. In low-grade dysplasia and high-grade dysplasia, there was no mucin present in the dysplasia epithelium. The difference between the two groups was statistically significant (P<0.01). The positive rate of Ki-67 in the epithelial reactive hyperplasia (>10%) was 81.0% (51/63), and the positive cells were mainly located in the neck and middle parts of the mucosal glands (58/63, 92.1%). In the low-grade dysplasia group, the positive rate of Ki-67 (>10%) was 90.7%(49/54); the positive cells were mainly located in the upper mucosa (33/54, 61.1%), showing a banded distribution pattern; in the high-grade dysplasia group, the positive rate (>10%) was 95.2%(60/63), and the positive cells were mainly located in the whole mucosa (49/63, 77.8%), showing a diffuse/diffuse scattered distribution pattern. The three groups had statistically different rates and distribution patterns of Ki-67 expression (P<0.01). Conclusion: The gastric epithelial reactive hyperplasia and dysplasia can be differentiated using clinicopathological features, AB-PAS staining and Ki-67 expression pattern.

Establishment of a rat model of laryngeal precancerous lesions induced by 4NQO smearing

Objective: To establish a rat model for laryngeal precancerous lesions histologically and pathologically comparable to the human counterpart. Methods: Thirty-six Wistar rats were randomly divided into experimental group and control group, with 18 rats in each group, and 1% 4-nitroquinoline-1-oxide (4NQO) solution and saline were respectively applied to the laryngeal mucosas of rats in two groups. During subsequent 20 weeks, the changes of laryngeal mucosas were regularly observed with naked eyes and endoscope and lesions were determined by histology. SPSS 22.0 software was used for statistical analysis. Results: The food intake, water intake and body weight of the rats in the experimental group were lower than those in the control group, with statistically significant differences (all P<0.05). White plaque, superficial ulcer, erosion and miliary particles were present in the larynxes of rats in the experimental group, with histological manifestations of atypical hyperplasia or carcinoma in situ, and normal epitheliums were shown in the control group. The number of Ki67 positive cells in the laryngeal mucosas of rats in the experimental group at the 4 th, 8 th, 12 th, 16 th, and 20 th weeks were 13.5±2.4, 35.6±5.8, 53.4±8.3, 78.8±11.6, 80.6±12.4, respectively, no Ki67 positive cells were found in the control group at individual time points, and the differences were statistically significant (t=9.74, 10.63, 11.14, 11.77, 11.26, respectively, all P<0.01). Conclusion: 4NQO can credibly cause rats laryngeal precancerous lesions, which morphologically and histologically mimic laryngeal carcinnogenesis. This method is practical, easy and reliable to prepare the animal model of laryngeal precancerous lesions.

Dietary butyrate, lauric acid and stearic acid improve gut morphology and epithelial cell turnover in weaned piglets.

This study was to evaluate the effects of the supplementation of saturated fatty acids with different chain lengths on growth performance, intestinal morphology, epithelial cell proliferation, differentiation and apoptosis in weaned piglets. Thirty-two weaned piglets (Duroc × Landrace × Yorkshire, BW = 7.81 ± 0.26 kg) were weaned at 21 d and randomly assigned to 1 of 4 experimental treatments: (1) a basal diet (control); (2) control + 0.3% butyrate (BT); (3) control + 0.3% lauric acid (LA); (4) control + 0.3% stearic acid (SA). All piglets were then slaughtered for tissue sampling after having been fed experimental diets for 28 d after weaning. Supplementation of BT increased the gain-to-feed ratio (G:F) (P < 0.05) compared to piglets fed the control diet from 14 to 28 d. In addition, the villus height (VH) to crypt depth (CD) ratio (VH:CD ratio) of the ileum were higher in the BT and LA diets than that of the control diet (P < 0.05). The SA-supplemented diet increased ileal VH (P < 0.05), whereas the BT-supplemented diet increased jejunal CD (P < 0.05). Compared to the control, diets supplemented with BT, LA, or SA all tended to increase jejunal proliferation (Ki67/crypt positive cells) (P = 0.190); diets supplemented with BT or SA significantly increased the number of ki67-positive cells in the ileal crypt (P < 0.05). Furthermore, in the jejunum, the protein expression of activated caspase 3 and villin were increased in piglets fed BT, LA, or SA diets compared to those on the control diet (P < 0.05). In the ileum, compared with the control diet, the BT diet tended to increase the protein level of mammalian phosphorylation target of rapamycin (p-mTOR, P < 0.10); LA or SA diets significantly increased p-mTOR protein expression (P < 0.05). These results show that dietary supplementation of BT, LA, or SA promotes jejunal cell renewal in weaned piglets. At the same time, increased proliferation of ileal crypt cells by promoting p-mTOR expression has beneficial effects on ileal morphology in weaned piglets.

Lactobacillus derived from breast milk facilitates intestinal development in IUGR rats.

The intestinal microbiota contributes to infant's intestine homeostasis. This study aimed to analyse how probiotics derived from breast milk promote infant intestinal development in rat pups. The effect of potential probiotics derived from breast milk on development of intrauterine growth retardation (IUGR) newborn rats' intestine was investigated. Limosilactobacillus oris ML-329 and Lacticaseibacillus paracasei ML-446 exhibited good hydrophobicity percentages (p < 0.05). ML-446 showed a significant effect on intestinal length and weight (p < 0.05). Meanwhile, the villus height of the IUGR newborn rats fed with ML-329 was significantly higher compared with those fed with Lacticaseibacillus rhamnosus GG (p < 0.05). Moreover, ML-329 and ML-446 both significantly stimulated the proliferation and differentiation of intestinal epithelial cells by increasing the number of ki67-positive cells, goblet cells, and lysozyme-positive Paneth cells (p < 0.05) through Wnt and Notch pathway. The proliferation and differentiation stimulating effects of ML-329 and ML-446 on IECs in the jejunum, ileum, and colon were mediated by activating the Wnt pathway with increased expression of wnt, lrp5, and β-catenin genes and accumulation of β-catenin, and by downregulating the Notch signalling pathway with decreased expression of the activated notch protein. Lactobacillus could facilitate IUGR rat pups' intestinal development and enhance the proliferation of Paneth cells and goblet cells. These findings provide further insights into promotion of the intestinal development by breast milk-derived beneficial microbes in early life of the IUGR newborn rats.

Transcriptome-metabolome-wide association study (TMWAS) in rats revealed a potential carcinogenic effect of DEHP in thyroid associated with eicosanoids

The incidence of thyroid cancer (TC) has increased considerably in the last few decades. Environmental factors, including plasticizers, are recognized as potential risks leading to thyroid cancer in humans. In this study, we used a transcriptome-metabolome-wide association study to find the unidentified carcinogenic mechanism of di-2-ethylhexyl phthalate (DEHP) in thyroid and biomarkers for non-invasive diagnosis. Rats were treated with different doses of DEHP (0, 0.3, 3, 30, 150 mg DEHP/kg bw/day) for 13 weeks. Then, the thyroids were processed for Ki67 staining and RNA-seq. Also, 17-h urine samples were collected for high-resolution metabolomics analysis. After a high dose of DEHP exposure, the terminal body weights and the thyroid and parathyroid glands weights were not altered. However, the liver weights and numbers of Ki67-positive cells were increased. Further, multivariate statistical analysis revealed that metabolic shifts were considerably altered above 30 mg DEHP/kg bw/day. In RNA-seq analysis, some cancer-related genes were altered, including 18 upregulated and 9 downregulated transcripts. These cancer transcripts and whole metabolome data were integrated to uncover thyroid cancer-related metabolic pathways, which revealed that cancer-related transcripts had a network structure linked to eicosanoids such as leukotriene D4 and prostaglandin. In brief, our study demonstrated that DEHP can induce thyroid hyperplasia through the eicosanoid-associated pathway, providing further insight into the mechanism of DEHP-associated thyroid cancer.

Effect of fire needling on imiquimod induced psoriasis-like lesion and STAT3 pathway in mice

To observe the effect of fire needling on psoriasis-like lesion and the signal transducer and activator of transcription 3 (STAT3) pathway in mice and compare the therapeutic effect between different interventions of fire needling therapy (surrounding technique of fire needling, fire needling at "Dazhui" [GV 14] and "Zusanli" [ST 36]). Thirty male BALB/c mice were randomized into a blank group, a model group, a dexamthasone group, a surrounding technique group and an acupoint group, 6 mice in each one. Except the blank group, the mice in the rest groups were established as psoriasis-like lesion model by topical application with imiquimod cream, once daily, consecutively for 8 days. From day 4 to day 8, in the dexamthasone group, gastric infusion with 0.2 mL dexamthasone was administered, once daily. On day 4, 6 and 8, in the surrounding technique group, fire needling was exerted around the skin lesion; and fire needling was applied to "Dazhui" (GV 14) and "Zusanli" (ST 36) in the acupoint group, once a day. The changes in skin lesion on the dorsal parts of mice were observed in each group to score the psoriasis area and severity index (PASI). Using HE staining, the dermal morphological changes and epidermal thickness were observed in the mice of each group. The positive expression of proliferating cell-associated antigen Ki-67 was determined by immunofluorescence. Immunohistochemistry method was used to determine the expressions of , and T cells of skin tissue in each group. Using real-time PCR, the expressions of interleukin (IL)-17, IL-22, tumor necrosis factor α(TNF-α) mRNA were determined. Western blot method was adopted to determine the protein expressions of STAT3 and p-STAT3 in skin tissue in each group. Compared with the blank group, the scores of each item and the total scores of PASI, as well as the epidermal thickness were all increased in the mice of the model group (P<0.01). Except for the erythema scores of the dexamethasone group and the surrounding technique group, the scores of each item and the total scores of PASI, as well as the epidermal thickness were all decreased in each intervention group as compared with the model group (P<0.01). The infiltration scores and the total scores in the dexamethasone group and the acupoint group were lower than those in the surrounding technique group respectively (P<0.01, P<0.05). In comparison with the blank group, Ki-67 positive cell numbers and the numbers of , and T cells in skin tissue were increased in the mice of the model group (P<0.01). Ki-67 positive cell numbers and the numbers of , and T cells were reduced in each intervention group as compared with the model group (P<0.01), and the numbers of and T cells in the acupoint group were less than the surrounding technique group (P<0.01). Compared with the blank group, the mRNA expressions of IL-17, IL-22 and TNF-α and the ratio of p-STAT3 to STAT3 were all increased in the model group (P<0.01). The mRNA expressions of IL-17, IL-22 and TNF-α and the ratio of p-STAT3 to STAT3 were all decreased in each intervention group as compared with the model group (P<0.01, P<0.05). The mRNA expressions of IL-17, IL-22 and TNF-α in the acupoint group, as well as mRNA expression of IL-17 in the surrounding technique group were all lower than the dexamethasone group (P<0.01), while, the mRNA expression of IL-22 in the acupoint group was lower than the surrounding technique group (P<0.01). Fire needling therapy improves skin lesion severity in imiquimod induced psoriasis-like lesion of the mice, which is probably related to the inhibition of STAT3 pathway activation and the decrease of Th17 inflammatory factors expression. The systemic regulation of fire needling at "Dazhui" (GV 14) and "Zusanli" (ST 36) is superior to the local treatment.

Insulin and liraglutide attenuate brain pathology in diabetic mice by enhancing the Wnt/β-catenin signaling pathway.

Insulin and liraglutide have been demonstrated to control blood glucose and exert neuroprotective effects. However, the impact of liraglutide or insulin alone or in combination on brain pathology in type 1 diabetes mellitus (T1DM) and their underlying mechanisms are unclear. In the present study, diabetes mellitus (DM) was induced via intraperitoneal injection of streptozotocin in mice and subsequently mice were treated with insulin, liraglutide, a combination of the two drugs or saline. Changes in body weight and blood glucose were assessed weekly. The pathological changes in the brain tissue and the apoptosis of neurons were assessed using H&E staining and TUNEL staining. The mRNA and protein expression levels of apoptosis-related proteins were detected using reverse transcription-quantitative PCR (RT-qPCR) and western blotting, respectively. Moreover, Ki67 protein expression was analyzed using immunohistochemistry and the mRNA and protein expression levels of Wnt/β-catenin signaling pathway-related proteins were examined using RT-qPCR and western blotting, respectively. The results of the present study suggested that DM mice developed hyperglycemia and weight loss and also exhibited significantly increased neural cell apoptosis and significantly reduced numbers of Ki67-positive cells. Liraglutide significantly decreased blood glucose levels in DM mice, whereas both insulin and the combination of the two drugs failed to control blood glucose well. Insulin, liraglutide and their combination also failed to control body weight well, but significantly attenuated brain pathological changes and activation of the pro-apoptotic proteins Caspase-3 and Bax, which may have resulted in the significant increase in the expression levels of Wnt/β-catenin signaling pathway-associated molecules such as Wnt3a and S9-pGSK-3β. Liraglutide also promoted the protein expression of the neurogenesis marker of Ki67 and the antiapoptotic factor Bcl-2. These results suggested that insulin and liraglutide may improve brain damage via upregulation of the Wnt/β-catenin signaling pathway and could be of therapeutic relevance for improvement of cognitive impairment in patients with DM.

PSV-13 Lawsonia Intracellularis Increases Cell Proliferation in Swine Enteroids in Vitro

Abstract Lawsonia intracellularis is an enteric pathogen that causes thickening of the intestinal mucosa layer resulting in malnutrition, diarrhea, and poor weight gain in swine. L. intracellularis increases intestinal cell proliferation in vivo. Swine enteroids are 3-dimensional in vitro models that can be used to study bacterial infections. The objective of this study was to evaluate cellular proliferation in swine enteroids infected by L. intracellularis. Ki-67 is a protein expressed by cells that are actively proliferating and is often used to document changes in proliferation. Swine enteroids were cultured in monolayers using transwells, infected with L. intracellularis (PHE/MN1-00), and harvested on 5, 7, and 14 d post infection (dpi). Enteroid monolayers were fixed in 4% formaldehyde overnight, and immunofluorescence for Ki-67 and L. intracellularis were performed. Experiments were replicated 3 times. Immunofluorescent staining using L. intracellularis antibodies demonstrated infection of enteroids. Cell proliferation was assessed by counting the number of Ki-67 positive cells under 20x magnification and comparing to the total number of cells as stained by 4′,6-diamidino-2-phenylindole (DAPI) in infected and non-infected enteroids. Cell counts were analyzed using paired t-test in GraphPad Prism comparing infected enteroids with uninfected enteroids for each dpi time. Cell proliferation between non-infected and infected enteroids were not different at 5 dpi (0.014±0.007 vs. 0.044±0.021, P = 0.21) or 14 dpi (0.101±0.10 vs. 0.061±0.034, P = 0.46). However, at 7 dpi, infected enteroids showed greater cell proliferation (0.67±0.056 vs. 0.078±0.58, P = .047). In infected pigs, it was reported that increased cell proliferation is observed at 7 dpi. In vitro swine enteroids reproduce the proliferative changes observed in vivo, validating their use as a model for L. intracellularis infection.

The Importance of Ki67, Glial Fibrillar Acidic Protein and Vitamin D Receptor in the Central Nervous System of Postmortem Suicide Cases

Death by suicide is a tragic public health problem. Major depressive patients constitute some of these deaths. Although the etiology of major depression has not been clarified, there are studies suggesting that the hippocampus and caudate nucleus may be effective. In this study, it was aimed to compare vitamin D receptor (VDR), Ki67 and glial fibrial acidic protein (GFAP) antibodies in the brain tissues (hippocampus and caudate nucleus) of untreated major depression patients who died because of suicide with the control group. Left hippocampus and caudate nuclei of seven patients with untreated major depression who died of suicide in a one-year period and six psychiatrically normal patients were taken during autopsy. The hippocampus was stained with VDR, GFAP and Ki67 antibodies, and the caudate nucleus was stained with VDR and GFAP antibodies. There was no significant difference in the number of Ki67-positive cells in the anterior and posterior dentate gyrus between the major depression and control groups (P > 0.05). In the major depression group, a negative correlation was reported between the number of Ki67-positive cells in the anterior dentate gyrus and age (r: 0.867, P < 0.05). In the anterior dentate gyrus, posterior dentate gyrus and caudate nucleus, there was no significant difference between the suicide and control groups in the fraction of the immunoreactive area with GFAP antibodies (P > 0.05). While the percentage of immunoreactive granular cells for vitamin D receptor in the anterior dentate gyrus was significantly higher than the control group (P <0.05), no significant difference was reported in the posterior dentate gyrus. Vitamin D receptor immunoreactive neuron positivity in the caudate nucleus was not significantly different between the major depression and control groups (P > 0.05). In untreated major depression, additional immunohistochemical changes are observed in the anterior hippocampus, and the vitamin D receptor increases in the granular cells as a protective factor.

Cytokine-Induced JAK2-STAT3 Activates Tissue Regeneration under Systemic or Local Inflammation.

We investigated the immune response mechanisms under systemic and local inflammation using mouse models whereby lipopolysaccharide (LPS) was administered intraperitoneally to induce systemic inflammation, and epicutaneous sensitization with ovalbumin was used to induce local inflammation. LPS increased the immune cell infiltration in the cardiac muscle near the aorta, alveoli, hepatic sinusoid, renal interstitium, and the submucosal layer of the duodenum. Similarly, ovalbumin increased the abundance of macrophages in the skin. Both LPS and ovalbumin induced NF-κB p65 and IκBα phosphorylation, as well as the expression of NF-κB target genes (TLR4, IL6, and TNFα). Additionally, both LPS and ovalbumin led to an increase in the absolute IL-1β, IL-6, and TNFα serum levels and cytokine-related janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) phosphorylation. Moreover, the activated JAK2/STAT3 signaling increased the number of Ki67-positive cells (proliferating cells) and development pathway target gene expression (regeneration) in the inflammation models. In conclusion, LPS and ovalbumin increase immune cell infiltration in tissues, NF-κB activation, cytokine levels in serum, cytokine-stimulated JAK2/STAT3 signaling, and tissue regeneration.

Hippocampal Over-Expression of Cyclooxygenase-2 (COX-2) Is Associated with Susceptibility to Stress-Induced Anhedonia in Mice.

The phenomenon of individual variability in susceptibility/resilience to stress and depression, in which the hippocampus plays a pivotal role, is attracting increasing attention. We investigated the potential role of hippocampal cyclooxygenase-2 (COX-2), which regulates plasticity, neuroimmune function, and stress responses that are all linked to this risk dichotomy. We used a four-week-long chronic mild stress (CMS) paradigm, in which mice could be stratified according to their susceptibility/resilience to anhedonia, a key feature of depression, to investigate hippocampal expression of COX-2, a marker of microglial activation Iba-1, and the proliferation marker Ki67. Rat exposure, social defeat, restraints, and tail suspension were used as stressors. We compared the effects of treatment with either the selective COX-2 inhibitor celecoxib (30 mg/kg/day) or citalopram (15 mg/kg/day). For the celecoxib and vehicle-treated mice, the Porsolt test was used. Anhedonic (susceptible) but not non-anhedonic (resilient) animals exhibited elevated COX-2 mRNA levels, increased numbers of COX-2 and Iba-1-positive cells in the dentate gyrus and the CA1 area, and decreased numbers of Ki67-positive cells in the subgranular zone of the hippocampus. Drug treatment decreased the percentage of anhedonic mice, normalized swimming activity, reduced behavioral despair, and improved conditioned fear memory. Hippocampal over-expression of COX-2 is associated with susceptibility to stress-induced anhedonia, and its pharmacological inhibition with celecoxib has antidepressant effects that are similar in size to those of citalopram.