Alcian Blue-periodic acid-Schiff Research Articles

Xiaojianzhong decoction prevents gastric precancerous lesions in rats by inhibiting autophagy and glycolysis in gastric mucosal cells

Gastric precancerous lesions (GPL) precede the development of gastric cancer (GC). They are characterized by gastric mucosal intestinal metaplasia and dysplasia caused by various factors such as inflammation, bacterial infection, and injury. Abnormalities in autophagy and glycolysis affect GPL progression, and their effective regulation can aid in GPL treatment and GC prevention. Xiaojianzhong decoction (XJZ) is a classic compound for the treatment of digestive system diseases in ancient China which can inhibit the progression of GPL. However, its specific mechanism of action is still unclear. To investigate the therapeutic effects of XJZ decoction on a rat GPL model and the mechanisms underlying its effects on autophagy and glycolysis regulation in GPLs. Wistar rats were randomly divided into six groups of five rats each and all groups except the control group were subjected to GPL model construction for 18 wk. The rats' body weight was monitored every 2 wk starting from the beginning of modeling. Gastric histopathology was examined using hematoxylin-eosin staining and Alcian blue-periodic acid-Schiff staining. Autophagy was observed using transmission electron microscopy. The expressions of autophagy, hypoxia, and glycolysis related proteins in gastric mucosa were detected using immunohistochemistry and immunofluorescence. The expressions of the following proteins in gastric tissues: B cell lymphoma/Leukemia-2 and adenovirus E1B19000 interacting protein 3 (Bnip-3), microtubule associated protein 1 light chain 3 (LC-3), moesin-like BCL2-interacting protein 1 (Beclin-1), phosphatidylinositol 3-kimase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), p53, AMP-activated protein kinase (AMPK), and Unc-51 like kinase 1 (ULK1) were detected using western blot. The relative expressions of autophagy, hypoxia, and glycolysis related mRNA in gastric tissues was detected using reverse transcription-polymerase chain reaction. Treatment with XJZ increased the rats' body weight and improved GPL-related histopathological manifestations. It also decreased autophagosome and autolysosome formation in gastric tissues and reduced Bnip-3, Beclin-1, and LC-3II expressions, resulting in inhibition of autophagy. Moreover, XJZ down-regulated glycolysis-related monocarboxylate transporter (MCT1), MCT4, and CD147 expressions. XJZ prevented the increase of autophagy level by decreasing gastric mucosal hypoxia, activating the PI3K/AKT/mTOR pathway, inhibiting the p53/AMPK pathway activation and ULK1 Ser-317 and Ser-555 phosphorylation. In addition, XJZ improved abnormal gastric mucosal glucose metabolism by ameliorating gastric mucosal hypoxia and inhibiting ULK1 expression. This study demonstrates that XJZ may inhibit autophagy and glycolysis in GPL gastric mucosal cells by improving gastric mucosal hypoxia and regulating PI3K/AKT/mTOR and p53/ AMPK/ULK1 signaling pathways, providing a feasible strategy for the GPL treatment.

Rhein Exhibits Anti-Inflammatory Effects in Chronic Atrophic Gastritis via Nrf2 and MAPK Signaling.

Chronic atrophic gastritis is a premalignant lesion with a high risk of developing into gastric cancer. Rhein is a key active ingredient of several traditional Chinese medicines with multiple pharmacological effects. Nevertheless, the role of rhein in chronic atrophic gastritis is unclear. Helicobacter pylori infection was used to establish chronic atrophic gastritis in a mouse model. Murine gastric mucosa treated with saline or rhein was used in experiments. Hematoxylin and eosin staining and Alcian blue-periodic acid-Schiff staining were utilized for histopathological observation of murine gastric mucosa. The levels of proinflammatory factors were detected by enzyme-linked immunosorbent assay, and oxidative stress-associated markers were detected by commercially available assay kits. Western blotting was used for measuring the levels of nuclear factor, erythroid 2-like bZIP transcription factor 2, and mitogen-activated protein kinase signaling-related proteins. Rhein mitigated the gastric mucosal injury and suppressed inflammation and oxidative stress in H. pylori-infected chronic atrophic gastritis mouse models. Rhein inactivated mitogen-activated protein kinase and activated erythroid 2-like bZIP transcription factor 2 signaling in gastric mucosa of mice with chronic atrophic gastritis. Rhein exhibits anti-inflammatory and antioxidant effects in chronic atrophic gastritis via erythroid 2-like bZIP transcription factor 2 and mitogen-activated protein kinase signaling.

The morphological characteristics and modification of the oviducal glands adapted for long-term sperm storage and fertilization in Amphioctopus fangsiao

Sperm storage is an important reproductive strategy in Amphioctopus fangsiao. Sperm can be stored in the oviducal glands up to eight months before fertilization. However, studies on the underlying storage mechanisms remain limited. In this study, the morphological structures of the oviductal glands and the changes adapted for sperm storage and fertilization were investigated by Hematoxylin eosin (HE) staining, transmission electron microscopy (TEM), and Alcian blue periodic acid Schiff (AB-PAS) staining. Results revealed that the nucleo-cytoplasmic ratio of the peripheral gland cells decreased and the eosinophilic secretory granules (glycoproteins) appeared in the cytoplasm and lumen during sperm storage. The cytoplasm of the central gland appeared vacuolated and AB pH 2.5 (+) secretions (acid mucins) were dispersed in the cells. These secretions may pass through the central cavity into the oviducts and be involved in zygote packaging. The spermathecae are composed of columnar epithelial ciliated cells with secretory functions. PAS (+) glycoproteins were found in the lumen, providing a microenvironment conducive to sperm survival. Cilia in the oviducts, central cavity, and spermathecae may help with sperm delivery, storage, and release, and egg transport. This study enhances our understanding of the biological reproduction process in A. fangsiao.

Quantitative Analysis of Mucin Expression Using Combined Alcian Blue-Periodic Acid Schiff (AB-PAS) Stain and Combined High Iron Diamine-Alcian Blue (HID-AB) Stain and the Correlation With Histomorphological Score in Chronic Calculous Cholecystitis.

Introduction Chronic calculous cholecystitis is the most common and important contributing factor for cholecystectomy across the country, with a prevalence of 2-29%. Cholesterol supersaturated bile plays a major role in stone formation. It is very essential to identify the pathogenesis of stone formation in order to prevent its formation. This study is aimed to evaluate histomorphological features of chronic calculous cholecystitis and to quantitatively evaluate alteration in mucin expression using the combined Alcian blue-periodic acid Schiff (AB-PAS) stain and the combined high iron diamine Alcian blue (HID-AB) stain, to correlate with each other and also with biochemical features of gall stones. Methods A cross-sectional study of 64 chronic calculous cholecystitis were taken for histomorphological assessment and grading using Hematoxylin and Eosin (H&E) stainand Masson's trichrome stains.Expression of the type of mucins wasanalyzed using histochemical stains by a standardized scoring system. Results A significant positive correlation was observed between an increase in grades of inflammation and fibrosis with an increase in the quantity of sialomucin and neutral mucin in the deep layers of epithelium, and a significant negative correlation was observed between an increase in grades with a decrease in acidic mucin and sulfomucin of both superficial and deep epithelium except sulfomucin in fibrosis. No significant correlation was obtained with muscle thickness, adipose tissue deposition, and epithelial hyperplasia. A higher frequency of mixed-type stones was associated with severe inflammation. Conclusion Inflammation and fibrosis were strongly correlated with quantitative alteration and reversal of mucin composition in chronic cholecystitis; hence we conclude that these features play a significant role in the pathogenesis of stone formation. Using Combined AB(2.5pH)-PAS stain and Combined HID-AB(2.5 pH) stain to detect mucin hypersecretion and composition of altered mucin is relatively accurate and cost-effective rather than performing costly immunohistochemical (IHC) markers.

Effect of In Ovo Trace Element Supplementation on Immune-Related Cells of the Small Intestine of Post-hatched Broiler Chicken.

Pathological conditions and harmful drugs cause many gastrointestinal diseases in broiler chicken. The current study was conducted to investigate the effect of trace elements zinc (Zn) and selenium (Se) supplementation on histomorphology, immunological role, and functional activity of goblet cells (GCs) of the small intestine. The Alcian blue-periodic acid-Schiff (AB-PAS) was performed to assess the histomorphological changes in GCs, which revealed the regular dispersion with high electron density of GCs throughout the mucosal surface in the supplemented group. However, irregular dispersion with low electron density of GCs was present in the control group. The immunological functional role of GCs within the small intestine was examined by mucicarmine staining, immunohistochemistry, and immunofluorescence. The results showed a high mucin glycol protein secretion in the supplemented group, whereas limited mucin glycol protein secretion in the control group. Furthermore, the biological significance showed a high and low immunoreactivity of Muc2 and Muc13 in the supplemented and control groups, respectively. Immunofluorescence was used to confirm the immunosignaling of Muc2. Results revealed high immunosignaling of Muc2 at the apical part of the small intestine in the supplementation group, while low immunosignaling of Muc2 in the control group. Results suggest that trace element supplementation had significant effect on morphology and immunological role of GCs, which might be essential for immune function and health status of broiler chicken.

Mucin Level as a Potential Biomarker for Breast Cancer Diagnosis

Background: Breast cancer is the second leading cause of cancer death and a health problem worldwide. Secreted mucins are upregulated in ductal adenocarcinoma of the breast, however, the use of mucin as breast cancer biomarker has not been established before. This study aimed to determine the use of mucin level as a potential biomarker for breast cancer diagnosis. Materials and methods: This was a retrospective, cross-section study involving 40 women subjects with breast cancer. Mucin level was examined with a combination of Alcian blue/periodic acid Schiff (AB/PAS) technique applied to each specimen. The results obtained were statistically analyzed using SPSS.Results: Results of neutral mucin detection showed that among breast cancer subjects, 16 cases (40%) were neutral mucin score (+), 23 cases (57.5%) were neutral mucin score (++), and 1 case (2.5%) was neutral mucin score (+++). Meanwhile, 10 cases (25%) were acid mucin negative, 17 cases (42.5%) were acid mucin score (+), 11 cases (27.5%) were acid mucin score (++), and 2 cases (5%) were acid mucin score (+++). The most frequent type of mucin was the combination of acid and neutral mucin (30 cases; 75%) and neutral mucin were 10 cases (25%). Conclusion: Detection of mucin level can be used as an alternative technique for the diagnosis of breast cancer complementary to other types of special stains. Keywords: AB, PAS, breast cancer, histological grade, mucin level

Jatrorrhizine Alleviates DSS-Induced Ulcerative Colitis by Regulating the Intestinal Barrier Function and Inhibiting TLR4/MyD88/NF-κB Signaling Pathway.

Background Ulcerative colitis (UC), a kind of autoimmune disease with unknown etiology, has been troubling human physical and mental health. Jatrorrhizine (Jat) is a natural isoquinoline alkaloid isolated from Coptis Chinensis, which has been proved to have antibacterial, anti-inflammatory, and antitumor effects. Purpose The purpose is to explore the therapeutic effect of Jat on DSS-induced UC and the mechanism of action. Study Design. The UC mice model was induced by 3% DSS in drinking water. The mice were orally administered with Jat (40, 80, 160 mg/kg) for 10 days. Methods The changes in body weight, colon length, spleen wet weight index, disease activity index (DAI), colonic histopathology, and inflammatory factors of serum and colon tissue were analyzed to evaluate the severity of colitis mice. The colon mucus secretion capacity was analyzed by Alcian blue periodic acid Schiff (AB-PAS) staining. Furthermore, protein expressions such as TLR4, MyD88, p–NF–κB-p65, NF-κB-p65, COX-2, ZO-1, and Occludin were detected to elucidate the molecular mechanism of Jat on DSS-induced colitis model. Results The results showed that Jat could significantly alleviate the symptoms, colon shortening, spleen index, and histological damage and restore the body weight in DSS-induced colitis mice. Jat also suppressed the levels of inflammatory cytokines and upregulated the levels of anti-inflammatory cytokines. In addition, Jat repaired the intestinal barrier function by upregulating the level of colonic tight junction (TJ) proteins and enhancing the secretion of mucin produced by goblet cells. Furthermore, Jat could significantly suppress the expression of TLR4, MyD88, p–NF–κB-p65/NF-κB-p65, and COX-2 in colon tissue. Conclusion The results suggested that Jat plays a protective role in DSS-induced colitis by regulating the intestinal barrier function and inhibiting the TLR4/MyD88/NF-κB signaling pathway. This study, for the first time, demonstrates the therapeutic and protective effects of Jat on UC.

Baicalin Ameliorates DSS-Induced Colitis by Protecting Goblet Cells through Activating NLRP6 Inflammasomes

Objective Baicalin is an active compound found in many natural herbs and has been used to treat intestinal disorders such as diarrhea and colon cancer. In this study, we used a dextran sodium sulfate (DSS)-induced colitis mouse model to investigate baicalin's mechanisms in the treatment of colitis. Methods 3% DSS was administered through the drinking supply for 7 days to induce colitis followed by the administration of 5-aminosalicylic acid and baicalin at three different doses (25, 50, and 100 mg/kg, W/W) for an additional 7 days. Body weight, stool consistency, and colon length were recorded. Colon tissue was stained with hematoxylin and eosin (H&E) to be used for histopathological scoring. Cytokine levels of the colon tissue and serum were evaluated using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. mRNA expression and protein levels of tight junctions (TJs) were detected with qRT-PCR and Western blotting. Goblet cells and the mucosal layer of the colon were visualized by Alcian Blue/periodic acid-Schiff (AB/PAS) staining. Mucin 2 (MUC2) was evaluated in both mRNA expression and protein levels. Nod-like receptor pyrin domain-containing protein 6 (NLRP6) inflammasomes were detected by immunohistochemistry and Western blotting. Results Treatment with baicalin significantly relieved colitis as evidenced by reversing both weight loss and colon length shortening. In addition, baicalin inhibited inflammation by reducing proinflammatory cytokines and protected the intestinal barrier by upregulating tight junction proteins. Moreover, goblet cell count and intestinal mucosa thickness were both significantly increased after baicalin treatment. Giving baicalin could upregulate the expression of NLRP6 and interleukin (IL-18) both in mRNA and protein. Conclusion Baicalin ameliorates DSS-induced colitis by protecting goblet cells through activating NLRP6 inflammasomes.

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.

Comparison of Different Histological Staining Methods for Detection of Helicobacter pylori Infection in Gastric Biopsy.

Background: Helicobacter pylori gastritis affects two-thirds of the world's human population. Among various invasive and non-invasive tests, histology play a very important role in detecting H. pylori in gastric biopsies. In histology, for detection of H. pylori, we use different histological staining techniques like routine haematoxylin & eosin (H&E) stain, Giemsa stain, Gimenez stain, and periodic acid Schiff - Alcian blue (PAS-AB) stain.Objective: Aim of our study was to evaluate these different histopathological staining techniques for detecting H. pylori in gastric mucosal biopsies and to determine the sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of H&E stain, Gemenez stain and PAS-AB in the detection of H. pylori in gastric biopsies using Giemsa stain as the reference standard.Materials and Methods: It was a prospective descriptive study design of 45 gastric biopsies of patients having gastritis. This study was conducted at Vivekananda Polyclinic and Institute of Medical Sciences over a period of one year, from March 2021 to February 2022. From each formalin-fixed paraffin-embedded block, four glass slides were prepared and stained with H&E stain, Giemsa stains, Gemenez stain, and PAS-AB stain to detect the presence/absence of H. pylori in gastric biopsies. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy were assessed.Results: Various staining techniques for detecting H. pylori in gastric mucosal biopsies were compared. In reference to Giemsa stain results, statistical analysis indicates that the diagnostic accuracy of the Gimenez stain, H&E stain, and PAS-AB stain were 95.6%, 91.1%, and 84.4% respectively. Gimenez stain is confirmed to be better than H&E stain and PAS-AB stain to detect H. pylori in 45 gastric biopsies of patients having gastritis. PAS-AB stain is the worst stain to detect H. pylori in gastric biopsies.Conclusion: Gimenez stain has higher diagnostic accuracy than PAS-AB stain in the detection of H. pylori in gastric biopsy. In fact, Gimenez stain has high sensitivity, specificity and diagnostic accuracy as compared to traditional H&E stain while PAS-AB stain has lower sensitivity, specificity and diagnostic accuracy. Thus, Gimenez stain is also recommended for the detection of H. pylori in gastric biopsy.

Epstein–Barr virus-associated gastric adenosquamous carcinoma with concurrent gastric carcinoma with lymphoid stroma: a case report and review of the literature

BackgroundAdenosquamous carcinoma (ASC)with concurrent gastric carcinoma with lymphoid stroma (GCLS) are extremely rare tumors. There are only limited cases reported in the literature. Epstein–Barr virus (EBV) infection was found in the concomitant GCLS, but none in the ASC. Here, we report the first case of gastric cancer with EBV infection detected in both ASC and GCLS.Case presentationA 59-year-old man complained of intermittent upper abdominal pain. The gastric endoscopy revealed a type IIc tumor located in the gastric body near the fundus of the stomach. Histological examination of the gastric tumor showed the coexistence of ASC and GCLS. Both components were positive for EBV-encoded RNA (EBER) in situ hybridization. Neoplastic nests of the former were positive for p63, p40 and CK5/6. The glandular components showed positive acid mucus in the Alcian-blue periodic-acid-schiff (AB-PAS) staining. There was significant difference in the expression of epidermal growth factor receptor (EGFR) between adenocarcinoma and squamous carcinoma, but not in other proteins such as human epidermal growth factor receptor 2 (HER2), p53 and mismatch repair proteins. The role of EGFR signaling pathway needs to be further explored in the differentiation of squamous carcinoma in the gastric ASC. Finally, a diagnosis of early EBV associated gastric ASC with concurrent GCLS (pT1bN1) was made. The patient took a single-drug S1 periodically for half a year after the surgery and has been disease free during 8 months of medical follow-up.ConclusionsThis is the first case of EBV associated gastric ASC with concurrent GCLS, and pathologists and clinicians should recognize and pay attention to this type of tumor.

Intestinal mucosal barrier in functional constipation: Dose it change?

BACKGROUNDThe intestinal mucosal barrier is the first line of defense against numerous harmful substances, and it contributes to the maintenance of intestinal homeostasis. Recent studies reported that structural and functional changes in the intestinal mucosal barrier were involved in the pathogenesis of several intestinal diseases. However, no study thoroughly evaluated this barrier in patients with functional constipation (FC).AIMTo investigate the intestinal mucosal barrier in FC, including the mucus barrier, intercellular junctions, mucosal immunity and gut permeability.METHODSForty FC patients who fulfilled the Rome IV criteria and 24 healthy controls were recruited in the Department of Gastroenterology of China-Japan Friendship Hospital. The colonic mucus barrier, intercellular junctions in the colonic epithelium, mucosal immune state and gut permeability in FC patients were comprehensively examined. Goblet cells were stained with Alcian Blue/Periodic acid Schiff (AB/PAS) and counted. The ultrastructure of intercellular junctional complexes was observed under an electron microscope. Occludin and zonula occludens-1 (ZO-1) in the colonic mucosa were located and quantified using immunohistochemistry and quantitative real-time polymerase chain reaction. Colonic CD3+ intraepithelial lymphocytes (IELs) and CD3+ lymphocytes in the lamina propria were identified and counted using immunofluorescence. The serum levels of D-lactic acid and zonulin were detected using enzyme-linked immunosorbent assay.RESULTSCompared to healthy controls, the staining of mucus secreted by goblet cells was darker in FC patients, and the number of goblet cells per upper crypt in the colonic mucosa was significantly increased in FC patients (control, 18.67 ± 2.99; FC, 22.42 ± 4.09; P = 0.001). The intercellular junctional complexes in the colonic epithelium were integral in FC patients. The distribution of mucosal occludin and ZO-1 was not altered in FC patients. No significant differences were found in occludin (control, 5.76E-2 ± 1.62E-2; FC, 5.17E-2 ± 1.80E-2; P = 0.240) and ZO-1 (control, 2.29E-2 ± 0.93E-2; FC, 2.68E-2 ± 1.60E-2; P = 0.333) protein expression between the two groups. The mRNA levels in occludin and ZO-1 were not modified in FC patients compared to healthy controls (P = 0.145, P = 0.451, respectively). No significant differences were observed in the number of CD3+ IELs per 100 epithelial cells (control, 5.62 ± 2.06; FC, 4.50 ± 2.16; P = 0.070) and CD3+ lamina propria lymphocytes (control, 19.69 ± 6.04/mm2; FC, 22.70 ± 11.38/mm2; P = 0.273). There were no significant differences in serum D-lactic acid [control, 5.21 (4.46, 5.49) mmol/L; FC, 4.63 (4.31, 5.42) mmol/L; P = 0.112] or zonulin [control, 1.36 (0.53, 2.15) ng/mL; FC, 0.94 (0.47, 1.56) ng/mL; P = 0.185] levels between FC patients and healthy controls.CONCLUSIONThe intestinal mucosal barrier in FC patients exhibits a compensatory increase in goblet cells and integral intercellular junctions without activation of mucosal immunity or increased gut permeability.

Autocrine TGF-alpha is associated with Benzo(a)pyrene-induced mucus production and MUC5AC expression during allergic asthma

ObjectsBenzo(a)pyrene (BaP), an environmental pollutant, is present in high concentrations in urban smog and cigarette smoke and has been reported to promote high mucin 5AC (MUC5AC) expression. Epithelium-derived inflammatory cytokines are considered an important modulator of mucus oversecretion and MUC5AC overexpression. Here, we investigated whether the effect of BaP on MUC5AC overexpression was associated with cytokine autocrine activity in vivo and in vitro. MethodsIn vivo, BALB/c mice were treated with ovalbumin (OVA) in the presence or absence of BaP. Allergy-induced mucus production was assessed by Alcian Blue Periodic acid Schiff (AB-PAS) staining. The human airway epithelial cell line NCI-H292 was used in vitro. MUC5AC and transforming growth factor (TGF)-α mRNA levels were assessed with real-time quantitative PCR. The concentration of cytokines was measured by ELISA. The MUC5AC, p-ERK, ERK, p-EGFR and EGFR proteins were detected by Western blotting in cells or by immunohistochemistry in mouse lungs. Small-interfering RNAs were used for gene silencing. ResultsTGF-α was overproduced in the supernatant of NCI-H292 cells treated with BaP. Knockdown of TGF-α expression inhibited the BaP-induced increase in MUC5AC expression and subsequent activation of the EGFR-ERK signalling pathway. Knocking down aryl hydrocarbon receptor (AhR) expression or treatment with an ROS inhibitor (N-acetyl-L-cysteine) could relieve the TGF-α secretion induced by BaP in epithelial cells. In an animal study, coexposure to BaP with OVA increased mucus production, MUC5AC expression and ROS-EGFR-ERK activation in the lung as well as TGF-α levels in bronchoalveolar lavage fluid (BALF). Furthermore, the concentration of TGF-α in BALF was correlated with MUC5AC mRNA levels. Additionally, TGF-α expression was found to be positively correlated with MUC5AC expression in the airway epithelial cells of smokers. Compared with non-smoker asthma patients, TGF-α serum levels were also elevated in smoker asthma patients. ConclusionAutocrine TGF-α was associated with BaP-induced MUC5AC expression in vitro and in vivo. BaP induced TGF-α secretion by activating AhR and producing ROS, which led to activation of the EGFR-ERK pathway

Disordered Expression of Tight Junction Proteins Is Involved in the Mo-induced Intestinal Microenvironment Dysbiosis in Sheep.

To evaluate the molybdenum (Mo)-induced changes of intestinal morphology and the relationship of intestinal tight junction (TJ) proteins expression and intestinal barrier function, a total of 20 healthy sheep were randomly divided into five groups of four: 0, 5, 10, 20, and 50mg/kg BW/day Na2MoO4·2H2O were administrated in five groups named control group, Mo 5 group, Mo 10 group, Mo 20 group, and Mo 50 group, respectively. After 28days of Mo treatment, the duodenum, the jejunum, and the ileum tissue were collected. The histopathology and the developmental parameters were evaluated by hematoxylin-eosin staining. The intestinal epithelial cell DNA damage was detected by TdT-mediated dUTP nick end labeling assay. The intestinal glycoprotein and the goblet cells were analyzed by Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining and PAS staining, respectively. TJ proteins were determined by immunofluorescence technology. Results showed that excessive Mo significantly decreased the small intestinal villus height (VH), crypt depth (CD), VH/CD, and mucosal thickness (P < 0.05 or P < 0.01) while induced the damage of DNA in small intestinal epithelial cells. Moreover, excessive Mo injured intestinal barrier function by decreasing the percent of glycoprotein distribution area (P < 0.05) and the relative density of intestinal goblet cells (P < 0.05). Mo treatment induced decreased (P < 0.01) expression of Zonula Occludens-1, Occludin, and Claudin-1. In conclusion, excessive Mo interfered with the expression of TJ proteins, inhibited intestinal epithelial development, and further aggravated the intestinal barrier function damage, leading to disturbing the small intestinal microenvironment balance.

Life-stage dependent histological characterization of liver HIF-1α expression in the Indonesian shortfin eel (Anguilla bicolor bicolor McClelland, 1884)

The Indonesian shortfin eel (Anguilla bicolor bicolor McClelland, 1884) is catadromous fish. The developmental phase of the fish is influenced by the availability and type of feed, and dissolved oxygen levels (DO). Oxygen level fluctuations during migration from sea to fresh waters can cause hypoxia and trigger activation of the Hypoxia-inducible factor 1 alpha gene (HIF-1α). Therefore, undertanding HIF-1α expression is very important in understanding the life cycle and physiology of this organism. This study aimed to perform a histological comparison between the liver structure of eel in the elver eel phase to silver eel and compared the expression of HIF-1α in the liver. The eels were collected from Kulon Progo Regency, Segara Anakan in Cilacap, and Cangkringan in Sleman, respectively. The eel livers were prepared with standard paraffin method. The samples were stained with Hematoxylin-Eosin, Mallory Acid Fuchsin and Periodic Acid-Schiff Alcian Blue for histological observation. The HIF-1α protein expression was detected using immunohistochemistry. The results showed that the structure of liver elver, yellow, and silver eel shared similar tubular hepatocytes. Moreover, there was no significant differences in the parenchymal structure of liver of all stage of eel. Mucin levels were different in each life cycle phase. HIF-1α expression varied by eel phase, with the elver eel showing weak expression, yellow stage showing moderate expression, and silver eels displaying the highest levels of expression. We conclude that the highest expression of HIF-1α was found in the silver eel, which resides in freshwater environments and is found in freshwater while returning to the sea for reproduction.

Fructus Mume (Wu Mei) Attenuates Acetic Acid-Induced Ulcerative Colitis by Regulating Inflammatory Cytokine, Reactive Oxygen Species, and Neuropeptide Levels in Model Rats.

Ulcerative colitis (UC) is a chronic idiopathic inflammatory disorder of the large intestine. Fructus mume (FM), a natural food with nutritive and pharmaceutical value, has demonstrated therapeutic efficacy against UC. In this study, we investigated the protective effects and mechanisms of FM against UC. We induced UC in rats with 4% (v/v) acetic acid (AA), orally administered 0.7 or 0.325 g/kg FM and 0.3 g/kg sulfasalazine (SASP) for 7 days, and explored the responses the drugs elicited in the rats. We assessed the general conditions of the rats by the disease active index. We evaluated colon tissue damage macroscopically and by Hematoxylin & Eosin, Alcian Blue-periodic acid-Schiff, and Masson's staining, and explored the potential mechanisms of FM on inflammation, oxidative stress, and neuropeptides by measuring TNF-α, IL-6, IL-8, IL-10, MMP9, CXCR-1, SOD, GSH-px, MDA, ROS, SIRT3, SP, VIP, ghrelin, and 5-HT. FM treatment significantly attenuated colon damage and submucosal fibrosis compared with the model. It lowered serum proinflammatory TNF-α, IL-8, and colonic MMP9 and CXCR-1, and raised serum anti-inflammatory IL-10 levels. FM upregulated the antioxidant enzymes SOD, GSH-px, and SITR3 protein but inhibited ROS and MDA production. It downregulated colonic SP, VIP, ghrelin, and 5-HT. The beneficial effects of FM might be dose dependent. Around 0.7 g/kg FM and SASP displayed similar efficacy for treating AA-induced colitis in rats. Our results provide empirical evidence that FM protects against AA-induced UC in rats via anti-inflammatory and antioxidant mechanisms, and regulates neuropeptides; thus, FM may be a promising, safe, and efficacious alternative therapy for UC, if its efficacy can be confirmed in human trials.

An In Vivo Observational Histological Study of Peripheral Arterial Damage in Patients with Acute Limb Ischemia in SARS-CoV-2 Infection.

Thromboembolic events, such as acute limb ischemia, were reported worldwide in patients with COVID-19, suggesting that SARS-CoV-2 infection acts like a redoubtable prothrombotic factor in these patients. The aim of the study was to summarize the histopathological changes found in the arterial wall, intraarterial thrombus, and adjacent skeletal muscles. Considering the lack of evidence from in vivo studies, we performed observational histological research of peripheral arterial damage in patients with acute limb ischemia and SARS-CoV-2 infection. We investigated 22 patients with acute limb ischemia and SARS and harvested histopathological samples from those who agreed to this procedure. We performed histologic tissue harvesting during the revascularization procedure from the thrombosed area of the common femoral artery. Morphologic analysis was made on the hematoxylin-eosin (HE) stain. Special stains were also used—Elastica van Gieson (EvG) and Alcian Blue—Periodic Acid—Schiff (AB-PAS) and primary antibodies—CD45 and CD61. Our patients had significant risk factors for thrombus formation, since all of them had arterial hypertension, 81% had dyslipidemia, 73% were obese, 63% suffered from diabetes mellitus, and 45% were active smokers. The histological findings using immunohistochemistry (CD45 and CD68 reactions) or special and usual stains underlined the mechanism for ischemia production in SARS-CoV-2 patients. The main histological findings in our study were endothelial destruction and inflammation that were found in all analyzed structures.

METHYLTRANSFERASE SMYD2 MODULATES THERAPEUTIC EFFECTS OF EPIDERMAL GROWTH FACTOR IN INFLAMMATORY BOWEL DISEASE

Abstract BACKGROUND Inflammatory bowel disease (IBD) is a chronic and remitting inflammatory disorder of the gastrointestinal tract. A prior clinical trial demonstrated the ability of epidermal growth factor receptor (EGFR) ligand EGF to induce and maintain remission in ulcerative colitis. As a protein lysine methyltransferase, SET and MYND domain-containing protein 2 (SMYD2) regulates various cellular functions, including chromatin remodeling and tumorigenesis. A bioinformatic study has shown that EGFR might be a candidate for SMYD2-mediated methylation. In this research, we aimed to investigate whether SMYD2 depletion could synergize with EGFR-activating treatment to promote intestinal goblet cell regeneration in IBD. METHODS SMYD2 flox C57BL/6 (SMYD2fl/fl) and intestine epithelial cell-specific conditional knockout of SMYD2 (SMYD2ΔIEC) male mice (8 to10-week old) were given 3 cycles of DSS treatment, each cycle including 5 days of 2% DSS in drinking water followed by 16 days of normal drinking water. EGF (50 μg/kg/day) was administered via intraperitoneal injection once per day for 6 days during the third cycle of DSS treatment (from day 42 to 47 along with DSS administration). The disease activity index (DAI), including body weight loss, stool consistency and colorectal bleeding, was monitored and calculated daily. Histologic scoring was used to evaluate the extent of histopathologic change in middle colon tissue. Goblet cell differentiation and maturation was analyzed via Alcian Blue/periodic acid-Schiff (AB/PAS) staining and immunostaining. RESULTS EGF treatment significantly increased the number of goblet cells (AB/PAS staining positive cells) in both SMYD2fl/fl and SMYD2ΔIEC mice. However, in SMYD2ΔIEC mice, more mature goblet cells were observed. In contrast, in SMYD2fl/fl mice, most goblet cells were accumulated at the lower half of colon crypts with small mucin particles. As compared with SMYD2fl/fl mice receiving EGF treatment, SMYD2 depletion combined with EGF injection (6 days) failed to significantly improve body weight loss and DAI scoring during DSS treatment, as well as histologic scoring (including inflammatory cell infiltration and epithelial tissue damage). Immunostaining of goblet cell differentiation markers (including ATOH1, SPDEF and KLF4) revealed no significant difference between SMYD2fl/fl and SMYD2ΔIEC mice. CONCLUSION SMYD2 depletion combined with EGF treatment could promote goblet cell expansion and maturation in mouse DSS-induced chronic colitis. However, the mechanism by which SMYD2 depletion facilitates goblet cell maturation needs further investigation.

Lactobacillus paracasei from koumiss ameliorates diarrhea in mice via tight junctions modulation

ObjectivesProbiotics are gaining interest as alternative options for antibiotic or antiinflammatory drugs. Probiotics can affect the health of the host through metabolites and competitive inhibition adhesion of pathogenic microorganisms. Koumiss is an important part of the diet of Asian nomads, and is rich in a broad array of probiotics that can benefit the body. Mongolians have developed koumiss therapy to assist in the treatment of various diseases. In the present study, we investigate the beneficial effect of Lactobacillus paracasei, a strain isolated from koumiss, on a mouse model of diarrhea induced by Escherichia coli O8 (E. coli O8). MethodsProbiotics were isolated from Mongolian koumiss. The resistance of probiotics against acid, bile salts, gastric juice, and intestinal juice was evaluated. The mouse model of diarrhea was established by the intragastric administration of E. coli O8 after NaHCO3 treatment. L. paracasei was intragastrically administered before or after E. coli O8 exposure in mice. The plasma levels of diamine oxidase and zounlin were quantified using an enzyme-linked immunosorbent assay, and the integrity of the intestinal barrier and goblet cells of mice with diarrhea were observed using hematoxylin and eosin and Alcian blue periodic acid-Schiff staining. The expression of tight junction (TJ) proteins was detected by immunohistochemistry and Western blot. ResultsA total of five lactic acid bacteria and two yeast strains were isolated from koumiss, and L. paracasei was screened for animal experiments. Experimental results showed that L. paracasei could reduce the increase in diamine oxidase and zonulin caused by E. coli (P < 0.05); increase goblet cells and the expression of TJ proteins ZO-1, occludin, and claudin-1 (P < 0.05); increase the expression of mucin 2, oligomeric mucus/gel-forming (P < 0.05) protein; and reduce the level of inhibitor kappa B-alpha and myosin light-chain kinase. ConclusionsL. paracasei reduced the intestinal permeability, induced the expression of mucin 2, oligomeric mucus/gel-forming protein, and increased the number of goblet cells in mice by the upregulation of the expression of TJ proteins via the nuclear factor kappa B cells–myosin light-chain kinase signaling pathway.

Exosomes derived from adipose-derived stem cells alleviate cigarette smoke-induced lung inflammation and injury by inhibiting alveolar macrophages pyroptosis

BackgroundChronic obstructive pulmonary disease (COPD) is a frequently encountered disease condition in clinical practice mainly caused by cigarette smoke (CS). The aim of this study was to investigate the protective roles of human adipose-derived stem cells-derived exosomes (ADSCs-Exo) in CS-induced lung inflammation and injury and explore the underlying mechanism by discovering the effects of ADSCs-Exo on alveolar macrophages (AMs) pyroptosis.MethodsADSCs were isolated from human adipose tissues harvested from three healthy donors, and then ADSCs-Exo were isolated. In vivo, 24 age-matched male C57BL/6 mice were exposed to CS for 4 weeks, followed by intratracheal administration of ADSCs-Exo or phosphate buffered saline. In vitro, MH-S cells, derived from mouse AMs, were stimulated by 2% CS extract (CSE) for 24 h, followed by the treatment of ADSCs-Exo or phosphate buffered saline. Pulmonary inflammation was analyzed by detecting pro-inflammatory cells and mediators in the bronchoalveolar lavage fluid. Lung histology was assessed by hematoxylin and eosin staining. Mucus production was determined by Alcian blue-periodic acid-Schiff staining. The profile of AMs pyroptosis was evaluated by detecting the levels of pyroptosis-indicated proteins. The inflammatory response in AMs and the phagocytic activity of AMs were also investigated.ResultsIn mice exposed to CS, the levels of pro-inflammatory cells and mediators were significantly increased, mucus production was markedly increased and lung architecture was obviously disrupted. AMs pyroptosis was elevated and AMs phagocytosis was inhibited. However, the administration of ADSCs-Exo greatly reversed these alterations caused by CS exposure. Consistently, in MH-S cells with CSE-induced properties modelling those found in COPD, the cellular inflammatory response was elevated, the pyroptotic activity was upregulated while the phagocytosis was decreased. Nonetheless, these abnormalities were remarkably alleviated by the treatment of ADSCs-Exo.ConclusionsADSCs-Exo effectively attenuate CS-induced airway mucus overproduction, lung inflammation and injury by inhibiting AMs pyroptosis. Therefore, hADSCs-Exo may be a promising cell-free therapeutic candidate for CS-induced lung inflammation and injury.