Alcian Blue-periodic acid-Schiff Research Articles

WenTongGanPi decoction alleviates diarrhea-predominant irritable bowel syndrome by improving intestinal barrier

Ethnopharmacological relevanceWenTongGanPi Decoction (WTGPD) is a representative medical practice of the Fuyang School of Traditional Chinese Medicine (TCM), which originated from the classical Lu's Guizhi method. WTGPD places emphasis on the balance and functionality of yang qi, and is effective in treating TCM symptoms related to liver qi stagnation and spleen yang deficiency. In TCM, diarrhea-predominant irritable bowel syndrome (IBS-D) is often diagnosed as liver depression and spleen deficiency, and the use of WTGPD has shown significant therapeutic effect. However, the underlying mechanism of WTGPD treating IBS-D remains unclear. Aim of the studyTo explore the effect and mechanism of WTGPD in the treatment of IBS-D. Materials and methodsAn IBS-D model with liver depression and spleen deficiency was constructed by chronic immobilization stress stimulation and sennae folium aqueous gavage. The impact of WTGPD on IBS-D rats was evaluated through measurements of body weight, fecal water content, and abdominal withdrawal reflex (AWR). Intestinal permeability was assessed using hematoxylin-eosin (HE), alcian blue-periodic acid schiff (AB-PAS), immunofluorescence (IF) staining, and quantitative real-time PCR (qRT-PCR). The components of WTGPD were analyzed using UPLC-Q-TOF-MS. The underlying mechanisms were investigated through network pharmacology, transcriptomics sequencing, western blot (WB), molecular docking, and 16S rRNA sequencing. ResultsWTGPD treatment effectively alleviated diarrhea and abnormal pain in IBS-D rats (P < 0.05). It enhanced the intestinal barrier function by improving colonic structure and increasing the expression of tight junction proteins (P < 0.05). A total of 155 components were identified in WTGPD. Both network pharmacology and transcriptomics sequencing analysis highlighted MAPK as the key signaling pathway in WTGPD's anti-IBS-D effect. The WB results showed a significant decrease in p-p38, p-ERK and p-JNK expression after WTGPD treatment (P < 0.0001). Guanosine, adenosine and hesperetin in WTGPD may be involved in regulating the phosphorylation of p38, ERK and JNK. Additionally, WTGPD significantly enhanced microbial diversity and increased the production of colonic valeric acid in IBS-D rats (P < 0.01). ConclusionIn conclusion, our findings suggest that WTGPD can effectively alleviate IBS-D and improve intestinal barrier likely via inhibiting MAPK signal pathway and improving micobial dysbiosis.

Hydrogen-rich water alleviates constipation by attenuating oxidative stress through the sirtuin1/nuclear factor-erythroid-2-related factor 2/heme oxygenase-1 signaling pathway.

Constipation, a highly prevalent functional gastrointestinal disorder, induces a significant burden on the quality of patients' life and is associated with substantial healthcare expenditures. Therefore, identifying efficient therapeutic modalities for constipation is of paramount importance. Oxidative stress is a pivotal contributor to colonic dysmotility and is the underlying pathology responsible for constipation symptoms. Consequently, we postulate that hydrogen therapy, an emerging and promising intervention, can serve as a safe and efficacious treatment for constipation. To determine whether hydrogen-rich water (HRW) alleviates constipation and its potential mechanism. Constipation models were established by orally loperamide to Sprague-Dawley rats. Rats freely consumed HRW, and were recorded their 24 h total stool weight, fecal water content, and charcoal propulsion rate. Fecal samples were subjected to 16S rDNA gene sequencing. Serum non-targeted metabolomic analysis, malondialdehyde, and superoxide dismutase levels were determined. Colonic tissues were stained with hematoxylin and eosin, Alcian blue-periodic acid-Schiff, reactive oxygen species (ROS) immunofluorescence, and immunohistochemistry for cell growth factor receptor kit (c-kit), PGP 9.5, sirtuin1 (SIRT1), nuclear factor-erythroid-2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1). Quantitative real-time PCR and western blot analysis were conducted to determine the expression level of SIRT1, Nrf2 and HO-1. A rescue experiment was conducted by intraperitoneally injecting the SIRT1 inhibitor, EX527, into constipated rats. NCM460 cells were induced with H2O2 and treated with the metabolites to evaluate ROS and SIRT1 expression. HRW alleviated constipation symptoms by improving the total amount of stool over 24 h, fecal water content, charcoal propulsion rate, thickness of the intestinal mucus layer, c-kit expression, and the number of intestinal neurons. HRW modulated intestinal microbiota imbalance and abnormalities in serum metabolism. HRW could also reduce intestinal oxidative stress through the SIRT1/Nrf2/HO-1 signaling pathway. This regulatory effect on oxidative stress was confirmed via an intraperitoneal injection of a SIRT1 inhibitor to constipated rats. The serum metabolites, β-leucine (β-Leu) and traumatic acid, were also found to attenuate H2O2-induced oxidative stress in NCM460 cells by up-regulating SIRT1. HRW attenuates constipation-associated intestinal oxidative stress via SIRT1/Nrf2/HO-1 signaling pathway, modulating gut microbiota and serum metabolites. β-Leu and traumatic acid are potential metabolites that upregulate SIRT1 expression and reduce oxidative stress.

Mechanism of Salvia miltiorrhiza Bunge extract to alleviate Chronic Sleep Deprivation-Induced cognitive dysfunction in rats

BackgroundBidirectional communication between the gut microbiota and the brain may play an essential role in the cognitive dysfunction associated with chronic sleep deprivation(CSD). Salvia miltiorrhiza Bunge (Danshen, DS), a famous Chinese medicine and functional tea, is extensively used to protect learning and memory capacities, although the mechanism of action remains unknown. PurposeThe purpose of this research was to explore the efficacy and the underlying mechanism of DS in cognitive dysfunction caused by CSD. MethodsDS chemical composition was analyzed by UPLC-QTOF-MS/MS. Forty rats were randomly assigned to five groups (n = 8): control (CON), model (MOD), low- (1.35 g/kg, DSL), high-dose (2.70 g/kg, DSH) DS group, and Melatonin(100 mg/kg, MT) group. A CSD rat model was established over 21 days. DS's effects and the underlying mechanism were explored using the open-field test(OFT), Morris water-maze(MWM), tissue staining(Hematoxylin and Eosin Staining, Nissl staining, Alcian blue-periodic acid SCHIFF staining, and Immunofluorescence), enzyme-linked immunosorbent assay, Western blot, quantitative real-time polymerase chain reaction(qPCR), and 16S rRNA sequencing. ResultsWe demonstrated that CSD caused gut dysbiosis and cognitive dysfunction. Furthermore, 16S rRNA sequencing demonstrated that Firmicutes and Proteobacteria were more in fecal samples from model group rats, whereas Bacteroidota and Spirochaetota were less. DS therapy, on the contrary hand, greatly restored the gut microbial community, consequently alleviating cognitive impairment in rats. Further research revealed that DS administration reduced systemic inflammation via lowering intestinal inflammation and barrier disruption. Following that, DS therapy reduced Blood Brain Barrier(BBB) and neuronal damage, further decreasing neuroinflammation in the hippocampus(HP). Mechanistic studies revealed that DS therapy lowered lipopolysaccharide (LPS) levels in the HP, serum, and colon, consequently blocking the TLR4/MyD88/NF-κB signaling pathway and its downstream pro-inflammatory products(IL-1β, IL-6, TNF-α, iNOS, and COX2) in the HP and colon. ConclusionDS treatment dramatically improved spatial learning and memory impairments in rats with CSD by regulating the composition of the intestinal flora, preserving gut and brain barrier function, and reducing inflammation mediated by the LPS-TLR4 signaling pathway. Our findings provide novel insight into the mechanisms by which DS treats cognitive dysfunction caused by CSD.

Mechanism of n-butanol extract of Pulsatilla Decoction in treating ulcerative colitis by activating BMP signaling pathway

The study aimed to investigate the therapeutic effects of the n-butanol extract of Pulsatilla Decoction(BEPD) on ulcerative colitis(UC) via the bone morphogenetic protein(BMP) signaling pathway. C57BL/6 mice were divided into six groups: control, model, mesalazine, and BEPD low-, medium-, and high-dose groups. Except for the control group, the rest groups were treated with 3% dextran sulfate sodium(DSS) freely for seven consecutive days to establish the UC mouse model, followed by treatment with different concentrations of BEPD and mesalazine by gavage. The murine body weight and disease activity index(DAI) were recorded. After the mice were sacrificed, their colon tissues were collected for histological analysis. Alcian blue/periodic acid-Schiff(AB/PAS) staining was used to detect the number and mucus secretion status of goblet cells; immunohistochemistry was performed to measure the expression of ki67, cleaved caspase-3, mucin 2(Muc2), and matrix metalloproteinase-9(MMP9) in colon tissues; and immunofluorescence was used to analyze the expression of tight junction proteins in colon tissues, and enzyme linked immunosorbent assay(ELISA) was employed to quantify the levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-1β, and IL-6. Western blot was conducted to evaluate the expression of BMP pathway-related proteins in mouse colon tissues. Quantitative real-time PCR(qRT-PCR) was performed to measure the expression of genes related to goblet cell differentiation in mouse colon tissues. In addition, this study also examined the protective effect and underlying mechanism of BEPD-containing serum on lipopolysaccharide(LPS)-induced barrier damages in LS174T goblet cells in vitro. The results showed that BEPD significantly alleviated UC symptoms in mice, restored goblet cell diffe-rentiation function, promoted Muc2 secretion and tight junction protein expression, and suppressed inflammatory factor secretion while activating the BMP signaling pathway. Therefore, BEPD may exert its therapeutic effects on UC by activating the BMP signaling pathway, providing a new strategy for drug intervention in UC.

2'-Fucosyllactose restores the intestinal mucosal barrier in ulcerative colitis by inhibiting STAT3 palmitoylation and phosphorylation

2'-Fucosyllactose (2'-FL), the primary constituent of human milk oligosaccharides, has been identified as a potential regulator of inflammation in inflammatory bowel disease. Despite this recognition, the specific mechanisms through which 2'-FL alleviates ulcerative colitis (UC) remain ambiguous. This study seeks to investigate the potential anti-inflammatory properties of 2'-FL concerning intestinal inflammation and uncover the associated mechanisms. C57BL/6J mice were orally administered a daily dose of 500mg/kg 2'-FL for 11 consecutive days, followed by the induction of colitis using 3% (wt/vol) dextran sulfate sodium (DSS) for the final 6 days. Subsequently, a comprehensive range of techniques, including an Acyl-biotin exchange assay, fluorescein-isothiocyanate-labeled dextran assay, histopathology, ELISA, quantitative real-time PCR, Western blot, immunofluorescence staining, immunohistochemistry staining, Alcian blue-periodic acid schiff staining, TdT-mediated dUTP nick end labeling, transmission electron microscopy, iTRAQ quantitative proteomics, bioinformatics analysis, and the generation of signal transducer and activator of transcription 3 (STAT3) knockout mice, were employed to explore the relevant molecular mechanisms. Administration of 2'-FL significantly ameliorated DSS-induced colitis in mice and enhanced the integrity of the intestinal mucosal barrier. 2'-FL downregulated the phosphorylation of STAT3 and inhibited STAT3-related signaling pathways in colon tissues, which, in turn, reduced inflammatory responses. Interestingly, knockdown of STAT3 attenuated the protective effects of 2'-FL, highlighting that 2'-FL-mediated inflammatory attenuation is dependent on STAT3 expression. Additionally, 2'-FL could influence STAT3 activation by modulating the palmitoylation and depalmitoylation of STAT3. 2'-FL promotes the recovery of the intestinal mucosal barrier and suppresses inflammation in ulcerative colitis by inhibiting the palmitoylation and phosphorylation of STAT3.

1,2-bis(2,4,6-tribromophenoxy) ethane, a novel brominated flame retardant, disrupts intestinal barrier function via the IRX3/NOS2 axis in rat small intestine

Novel brominated flame retardants are widely used in electronics, textiles, furniture, and other products; they can enter the human body through ingestion and respiration and cause harm to the human body, and have been proven to have potential biological toxicity and accumulation effects. 1,2-bis(2,4,6-tribromophenoxy) ethane (BTBPE) is a widely used novel brominated flame retardant; however, there is a lack of research on its mechanism of toxicity, particularly that of intestinal toxicity. Currently, studies on the functionality of iroquois homeobox 3 (IRX3) are extremely limited. In our study, BTBPE was administered to Sprague-Dawley (SD) rats and rat small intestinal crypt epithelial cells (IEC6 cells) in vivo and in vitro, respectively, and hematoxylin and eosin (HE), immunohistochemical, Alcian blue-periodic acid-Schiff (AB-PAS), CCK8, acridine orange/ethidium bromide (AO/EB), fluorescent probes, qPCR, western blotting, and immunofluorescence analyses were performed. To explore the damage mechanism of BTBPE, we used siRNA to silence IRX3 and iNOs-IN-1 (yeast extract-peptone-wheat; YPW) to inhibit nitric oxide synthase 2 (NOS2). The results showed that BTBPE exposure caused inflammation and necroptosis in the jejunum and ileum, as well as destruction of the tight junctions and mucus layer. Moreover, BTBPE activated the IRX3/NOS2 axis both in vivo and in vitro. Silencing IRX3 or inhibiting NOS2 inhibits necroptosis and restores tight junctions in IEC6 cells. In conclusion, our study found that in the jejunum, ileum, and IEC6 cells, BTBPE exposure caused necroptosis and tight junction destruction by activating the IRX3/NOS2 axis. Blocking the IRX3/NOS2 axis can effectively inhibit necroptosis and restore tight junction. In addition, BTBPE exposure caused inflammation and loss of the mucous layer in the jejunum and ileum. Our study is the first to explore the mechanism of intestinal damage caused by BTBPE exposure and to discover new biological functions regulated by the IRX3/NOS2 axis, providing new research directions for necroptosis and tight junctions.

The effects of sodium butyrate (NaB) combination with soy saponin dietary supplementation on the growth parameters, intestinal performance and immune-related genes expression of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂)

Soy saponins are generally known to have negative effects on growth and the intestines of aquatic animals, and appropriate levels of sodium butyrate (NaB) may provide some mitigating effects. We investigated the effects of low and high levels of soy saponin and the protective effects of NaB (based on high level of soy saponin) on growth, serum cytokines, distal intestinal histopathology, and inflammation in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). The experiment included four groups: fishmeal group (FM, 0.00% saponin and 0.00% NaB), low saponin group (SL, 0.30% saponin and 0.00% NaB), high saponin group (SH, 1.50% saponin and 0.00% NaB) and high saponin with NaB group (SH–NaB, 1.50% saponin and 0.13% NaB). The results showed compared to FM, the final body weight (FBW) and weight gain (WG) were significantly higher and lower in SL and SH, respectively (P < 0.05). Compared to SH, the FBW and WG were significant higher in SH-NaB (P < 0.05). In the serum, compared to FM, the interferon γ (IFN-γ) and interleukin-1β (IL-1β) levels in SH were significantly increased (P < 0.05). Compared to SH, the IFN-γ level was significantly decreased in SH-NaB (P < 0.05). In the distal intestine, based on Alcian Blue-Periodic Acid-Schiff (AB-PAS) observation, the goblet cell/μm was significantly increased and decreased in the SL and SH, respectively, compared to FM. The intestinal diameter/plica height ratio in the SH was significantly higher than those in the FM, SL and SH-NaB (P < 0.05). The NO and ONOO− levels in the SH were significantly higher than that in FM and SL (P < 0.05). At the transcriptional level in the distal intestine, compared to FM, the mRNA levels of tumor necrosis factor (tnfα), il1β, interleukin-8 (il8) and ifnγ were significantly up-regulated in the SH (P < 0.05). Compared to the SH, tnfα, il8 and ifnγ were significantly down-regulated in the SH-NaB (P < 0.05). Compared to the FM, the mRNA levels of claudin3, claudin15, zo2 and zo3 were significantly up-regulated in the SL (P < 0.05). The mRNA levels of occludin, claudin3, claudin12, claudin15, zo1, zo2 and zo3 were significantly down-regulated in the SH compared to the FM (P < 0.05). Additionally, compared to the SH, the mRNA levels of occludin, claudin3, claudin12, claudin15, zo1, zo2 and zo3 were significantly up-regulated in the SH-NaB (P < 0.05). After the 7-day Vibrio parahaemolyticus challenge test, the survival was significantly higher and lower in the SL and SH, respectively, compared to FM (P < 0.05). Overall, low and high levels of soy saponins had positive and negative effects on growth, disease resistance, serum cytokines, and distal intestinal development and anti-inflammation, respectively, in hybrid grouper. NaB effectively increased disease resistance and improved distal intestinal inflammation in hybrid grouper, but the effects of NaB were mainly observed in improving distal intestinal tight junctions.

Cellular evidence of mucus cell immunological and differentiation characteristics in allogeneic crucian carp intestinal lamina propria

The allogeneic crucian carp is an important fish farm animal with a very different digestive system structure from that of mammals. The lamina propria of the fish intestine is also considered to be an important site of intestinal immunity in fish, but functional histological studies of the lamina propria of the allogeneic crucian carp intestine are still lacking. In this study, Identification of the ubiquitous lamina propria mucus cells in the lamina propria of the intestine by hematoxylin-eosin staining, and determination of the mucocytic properties, class, and distribution of these cells in each intestinal segment by Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining. The results show that type III mucus cells were abundant in the lamina propria of the foregut and midgut, while type II and type IV mucus cells predominate in the hindgut, possibly reflecting the distinct functions of these intestinal segments. Transmission electron microscopy dissected the differentiation of mucus cells in the lamina propria of the intestine at the ultrastructural level and investigated their morphology and distribution patterns in different intestinal segments, the findings revealed that lamina propria mucus cells perform rudimentary functions such as mucous secretion, phagocytosis, and degradation functions. Moreover, immunohistochemistry labeling with CD68 and LAMP1 revealed that numerous cells in the anterior, middle, and posterior intestines were positive for both proteins. Immunofluorescence double-labeling demonstrated that these cells highly co-expressed CD68 and LAMP1. Besides, the distribution and morphology of CD68+ and LAMP1+ cells were similar to those of AB-PAS positive cells and they accounted for the majority of parenchyma cells. Considering the above results, there were abundant cells with both mucous secretion and phagocytosis in the intestinal lamina propria of allogeneic crucian carp, which are a essential component of the intestinal immune process of allogeneic crucian carp.

Adaptation strategies of juvenile grass carp (Ctenopharyngodon idella) facing different dissolved oxygen concentrations in a recirculating aquaculture system

Fishes exposed to different dissolved oxygen concentrations over prolonged time periods have different susceptibility and adaptation mechanisms. In this study, healthy grass carp with an average weight of 214.6 ​± ​18.7 ​g were selected for long-term culture in recirculating aquaculture systems at three different dissolved oxygen concentrations, i.e., 3.00 ​mg/L [(2.57 ​± ​0.33) mg/L, DO2.57), 5.00 ​mg/L [(4.61 ​± ​0.11) mg/L, DO4.61], which was the control group, and 7.00 ​mg/L [(6.50 ​± ​0.48) mg/L, DO6.50]. Blood, gill, muscle, and liver samples were collected after 60 ​days. Using hematoxylin-eosin (HE), alcian blue periodic acid Schiff (AB-PAS) staining, determination of respiratory metabolism enzyme activity, and fluorescent quantitative PCR, the adaptability of juvenile grass carp facing different dissolved oxygen levels were explored. The results showed that the respiration rate increased, and hemoconcentration and hemoglobin (Hb), electrolyte and pH changed at DO2.57. Furthermore, gill lamellae became thinner and longer, the distance between them increased, the mucus on them decreased and the interstitial cell mass decreased. In addition, the relative expression of apoptosis-related genes bcl-2 and bcl-xl in gill tissue decreased, while caspase-3 and bax increased. In muscles and liver tissues, lactate dehydrogenase (LDH) activity and lactic acid content (LA) increased, pyruvate dehydrogenase (PDH) activity decreased, and genes related to the respiratory metabolic enzymes PDH-A4 and PDH-E1α also changed accordingly. The oxygen consumption rate decreased significantly (P ​< ​0.05). At DO6.50, the distance between gill lamellae, numbers of red blood cells (RBC) and the content of hemoglobin decreased wheras the mucus on gill lamellae increased. The activity of PDH, the expression of PDH-E1a in liver and oxygen consumption rate increased. Different dissolved oxygen concentrations affected the respiratory behavior, gill tissue structure, blood transport capacity and tissue enzyme activity of juvenile grass carp. In summary, juvenile grass carp showed different adaptation strategies when facing different dissolved oxygen concentrations over prolonged periods in recirculating aquaculture systems.

Lizhong decoction ameliorates pulmonary infection secondary to severe traumatic brain injury in rats by regulating the intestinal physical barrier and immune response.

The pathogenesis of pulmonary infection secondary to severe traumatic brain injury (sTBI) is closely related to damage to the intestinal barrier. Lizhong decoction (LZD) is a prominent traditional Chinese medicine (TCM) that is widely used in clinical treatment to regulate gastrointestinal movement and enhance resistance. Nevertheless, the role and mechanism of LZD in lung infection secondary to sTBI have yet to be elucidated. Here, we evaluate the therapeutic effect of LZD on pulmonary infection secondary to sTBI in rats and discuss potential regulatory mechanisms. The chemical constituents of LZD were analyzed by ultra-high performance liquid chromatography-Q Exactive-tandem mass spectrometry(UPLC-QE-MS/MS). The efficacy of LZD on rats with lung infection secondary to sTBI was examined by changes in brain morphology, coma time, brain water content, mNSS score, colony counts, 16S rRNA/RNaseP/MRP30kDa(16S/RPP30), myeloperoxidase (MPO) content and pathology of lung tissue. The concentration of fluorescein isothiocyanate(FITC)-dextran in serum and the contents of secretory immunoglobulin A (SIgA) in colon tissue were detected by enzyme-linked immunosorbent assay (ELISA). Subsequently, Alcian Blue Periodic acid Schiff (AB-PAS) was used to detect colonic goblet cells. Immunofluorescence (IF) was used to detect the expression of tight junction proteins. The proportions of CD3+ cell, CD4+CD8+ T cells, CD45+ cell and CD103+ cells in the colon were analyzed by flow cytometry (FC). In addition, colon transcriptomics were analyzed by Illumina mRNA-Seq sequencing. Real-time quantitative polymerase chain reaction (qRT‒PCR) was used to verify the genes associated with LZD alleviation of intestinal barrier function. Twenty-nine chemical constituents of LZD were revealed with UPLC-QE-MS/MS analysis. Administration of LZD significantly reduced colony counts, 16S/RPP30 and MPO content in lung infection secondary to sTBI rats. In addition, LZD also reduced the serum FITC-glucan content and the SIgA content of the colon. Additionally, LZD significantly increased the number of colonic goblet cells and the expression of tight junction proteins. Furthermore, LZD significantly decreased the proportion of CD3+ cell, CD4+CD8+ T cells,CD45+ and CD103+ cells in colon tissue. Transcriptomic analysis identified 22 upregulated genes and 56 downregulated genes in sTBI compared to the sham group. The levels of seven genes were recovered after LZD treatment. qRT‒PCR successfully validated two genes (Jchain and IL-6) at the mRNA level. LZD can improves sTBI secondary lung infection by regulating the intestinal physical barrier and immune response. Thees results suggested that LZD may be a prospective treatment for pulmonary infection secondary to sTBI.

Spermidine protects intestinal mucosal barrier function in mice colitis via the AhR/Nrf2 and AhR/STAT3 signaling pathways

Aryl hydrocarbon receptor (AhR) activation promotes intestinal barrier repair and enhances the gut mucosal barrier function in inflammatory bowel diseases (IBD). Spermidine is beneficial in several murine models of IBD and may affect AhR activity. However, the precise effects of spermidine on the intestinal barrier and AhR remain unclear. This study was designed to investigate whether spermidine affects AhR and gut barrier function in IBD models as well as, its underlying mechanism. We used dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mice, as well as, Caco2 cells incubated with TNF-α and IFN-γ to establish multiple IBD models, followed by spermidine intervention. Alcian blue/Periodic acid-Schiff (AB/PAS) staining, Fluorescein isothiocyanate (FITC)-dextran permeability assay, transepithelial electrical resistance (TER), tight junction protein (TJs) expression, and 16S rRNA scope in situ hybridization were performed to assess intestinal barrier function. AhR expression and the associated pathways were measured. AhR-targeted adeno-associated virus (AAV) and siRNA were used to explore the related molecular mechanisms. Spermidine significantly attenuated the increased intestinal permeability, decreased TER, abnormal distribution of TJs in colitis, and bacterial translocation from the gut tract. Additionally, it significantly increased AhR and Nrf2 expression and inhibited STAT3 phosphorylation. However, the protective effects of spermidine and the related alterations in pathway proteins were largely abolished by the specific inhibition of AhR. Our study demonstrated that spermidine rescues intestinal barrier defects in mice with colitis via the AhR-Nrf2 and AhR-STAT3 pathways, providing a potential therapeutic agent for IBD and other conditions associated with dysregulated gut barrier function.

The Effect of Maggot and Silkworm on the Growth and Intestinal Histological Structure of Wader Pari Fish (Rasbora lateristriata Bleeker, 1854)

Wader pari is one of the fish that is often found in Indonesian waters. One of the problems regarding fish farming is the problem of feed. This study aims to determine the effect of alternative feeding on the growth and histological structure of the Wader pari fish's intestine. The paraffin method was used in constructing intestinal histological preparations by Haematoxylin-Eosin (HE) and Periodic Acid Schiff-Alcian Blue (PAS-AB) staining. Observations were done using the DMRT test on various ANOVA, and the histological structure of the intestine was analyzed descriptively and quantitatively. The treatments used in this study were P1 (commercial feed), P2 (maggot feed), and P3 (mixed feed between maggot and silkworms). The study's results indicated that the higher the protein consumed by the Wader pari fish, the higher the growth. The three treatments that affected the growth of Wader pari sequentially are mixed feed, maggot feed, and commercial feed. The histological structure of the wader pari intestine is composed of tunica mucosa, tunica submucosa, tunica muscularis, and tunica serosa. Maggot and mixed feed can affect the histological structure of the Wader pari intestine compared to commercial feed. This can be seen in the results of the length of the villi and the distribution of the number of goblet cells.

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.