An Inulin-Type Fructan CP-A from Codonopsis pilosula Alleviated 5-Fluorouracil-Induced Intestinal Mucositis via the ERK/MLCK/MLC2 Pathway and Regulation of Gut Microbiota.

Intestinal mucositis (IM) is a common adverse effect of chemotherapy, limiting its clinical application. Codonopsis pilosula-derived CP-A (an inulin-type fructan) is an edible Chinese medicine with anti-inflammatory and gastrointestinal protective effects, which may be useful for treating IM. Here, we explored CP-A's role in ameliorating IM induced by 5-fluorouracil (5-FU) and investigated the underlying mechanism using in vitro experiments and rat models. Western blotting, immunohistochemistry (IHC), and real-time PCR (RT-PCR) analyses were used to assess protein expression related to the extracellular-regulated protein kinases (ERK)/myosin light chain kinase (MLCK)/myosin light chain 2 (MLC2) signaling pathway and tight junction proteins. Inflammatory factors were quantified using enzyme-linked immunosorbent assays (ELISAs), and 16S rRNA amplicon sequencing was employed for cecum content analysis. The results indicated that CP-A restored body weight and food intake and reversed histopathological changes in IM rats. Further, abnormal MLCK activation induced by 5-FU was attenuated by CP-A via the ERK/MLCK/MLC2 pathway. CP-A treatment improved tight junction protein levels and reduced inflammatory factor expression. Moreover, CP-A intervention regulated the intestinal microbiota community structure, increasing the abundance of Lactobacillus and decreasing the abundance of Shigella. In conclusion, CP-A mitigates 5-FU-induced IM by inhibiting the ERK/MLCK/MLC2 pathway, reducing the expression of inflammatory factors, improving the intestinal mucosal barrier, and regulating the intestinal microbial community. This study highlights CP-A's therapeutic potential in IM treatment and provides insights for future research.

Fermentation enhances the amelioration effect of bee pollen on Caco-2 monolayer epithelial barrier dysfunction based on NF-κB-mediated MLCK-MLC signaling pathway.

Intestinal barrier integrity is essential for normal nutrient digestion and absorption and disease resistance. This study aims to investigate how fermentation affects the ameliorative effect of bee pollen on the intestinal barrier dysfunction stimulated by interferon-γ and tumor necrosis factor (IFN-γ/TNF-α) cytokines. The results indicated that fermentation enhances the alleviating effect of bee pollen on intestinal barrier dysfunction (including elevated trans epithelial electrical resistance and decreased paracellular permeability). In addition, fermented bee pollen (FBP) significantly decreased (p < 0.05) the secretion levels of interleukin (IL)-6, IL-8, and IL-1ß and expression of cyclooxygenase (COX)-2 protein in intestinal barrier cells. Furthermore, fermentation improved the ability of bee pollen to up-regulate the expression of tight junction proteins including zonula occludens (ZO)-1, occluding, and claudin-1. Notably, FBP showed stronger ability to inhibit the expression of nuclear factor kappa-B (NF-κB) mediated myosin light chain kinase (MLCK) and myosin light chain (MLC) signaling pathway associated with phosphorylated proteins. Overall, our results indicated that fermentation enhances the protective effect of bee pollen on the intestinal barrier, and FBP has promising potential to be used as a novel functional food to protect the intestinal barrier.

Treatment with S-adenosylmethionine ameliorates irinotecan-induced intestinal barrier dysfunction and intestinal microbial disorder in mice.

This study aimed to investigate the protective effects of S-adenosylmethionine (SAM) on irinotecan-induced intestinal barrier dysfunction and microbial ecological dysregulation in both mice and human colon cell line Caco-2, which is widely used for studying intestinal epithelial barrier function. Specifically, this study utilized Caco-2 monolayers incubated with 7-ethyl-10-hydroxycamptothecin (SN-38) as well as an irinotecan-induced diarrhea model in mice. Our study found that SAM pretreatment significantly reduced body weight loss and diarrhea induced by irinotecan in mice. Furthermore, SAM inhibited the increase of intestinal permeability in irinotecan-treated mice and ameliorated the decrease of Zonula occludens-1(ZO-1), Occludin, and Claudin-1 expression. Additionally, irinotecan treatment increased the relative abundance of Proteobacteria compared to the control group, an effect that was reversed by SAM administration. In Caco-2 monolayers, SAM reduced the expression of reactive oxygen species (ROS) and ameliorated the decrease in transepithelial electrical resistance (TER) and increase in fluorescein isothiocyanate-dextran 4000 Da (FD-4) flux caused by SN-38. Moreover, SAM attenuated changes in the localization and distribution of ZO-1and Occludin in Caco-2 monolayers induced by SN-38 and protected barrier function by inhibiting activation of the p38 MAPK/p65 NF-κB/MLCK/MLC signaling pathway. These findings provide preliminary evidence for the potential use of SAM in treating diarrhea caused by irinotecan.

Forsythiaside A alleviates acute lung injury by inhibiting inflammation and epithelial barrier damages in lung and colon through PPAR-γ/RXR-α complex.

INTRODUCTION: Acute lung injury (ALI) is a lung disease characterized by inflammation and still requires further drug development. Forsythiaside A as the active compound of Forsythiae Fructus has the therapeutic potential for ALI. OBJECTIVE: To investigate the mechanism of forsythiaside A in treating ALI through PPAR-γ and its conjugate RXR-α based on gut-lung axis. METHODS: This study constructed in vitro and in vivo injury models using LPS and TNF-α. Forsythiaside A was used for the drug treatment, and RXR-α inhibitor UVI3003 was used to interfere with PPAR-γ/RXR-α complexes in the cells. HE staining was used for histopathological examination. Serum endotoxin contents were determined using limulus lysate kit. IHC staining and Western blot were conducted to assess the protein expressions. ELISA was applied to examine the content of pro-inflammatory cytokines in the cell supernatants. The protein interactions were analyzed via CO-IP. RESULTS: In vivo results showed that forsythiaside A regulated PPAR-γ/RXR-α and inhibited TLR4/MAPK/NF-κB and MLCK/MLC2 signal pathways, thus inhibiting inflammation and epithelial barrier damages of lung and colon in ALI mice induced by intratracheal LPS. PPAR-γ/RXR-α were promoted by forsythiaside A in lungs, whereas inhibited by forsythiaside A in colons. Additionally, in vitro results showed that forsythiaside A suppressed inflammation and epithelial barrier damages in macrophages and lung/colon epithelial cells, by manipulating PPAR-γ/RXR-α to suppress the LPS- and TNF-α-induced activation of TLR4/MAPK/NF-κB and NF-κB/MLCK/MLC2 signal pathways. Moreover, further mechanism study indicated that forsythiaside A showed a cell-specific regulatory effect on PPAR-γ/RXR-α complex. Specifically, the PPAR-γ/RXR-α protein interactions were promoted by forsythiaside A in LPS-induced macrophages RAW264.7 and TNF-α-induced lung epithelial cells A549, but inhibited by forsythiaside A in TNF-α-induced colon epithelial cells SW620. CONCLUSION: In the treatment of ALI, Forsythiaside A inhibited inflammation and epithelial barrier damages of lung and colon through its regulation on PPAR-γ/RXR-α complex.

Taxifolin ameliorates lipopolysaccharide-induced intestinal epithelial barrier dysfunction via attenuating NF-kappa B/MLCK pathway in a Caco-2 cell monolayer model.

Intestinal epithelial barrier dysfunction can cause several intestinal diseases. Flavonoids have been shown to be beneficial to the intestinal epithelial barrier function. However, the effects of taxifolin (TAX), a naturally occurring flavonoid, on the intestinal epithelial barrier function are unclear. Thus, the aims of this study were to investigate the protective effect and potential mechanism of TAX against lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction in a Caco-2 cell monolayer model. Our results showed that TAX increased the transepithelial electrical resistance (TEER) and decreased the fluorescein isothiocyanate (FITC)-dextran (4 kDa) flux in the damaged intestinal epithelial barrier. Meanwhile, TAX inhibited an LPS-induced decrease in mRNA and protein expression of tight junction (TJ) proteins (claudin-1, zonula occludens [ZO]-1, and occludin), and ameliorating the continuous distribution pattern disrupted of TJs. These results suggested that TAX ameliorated intestinal epithelial barrier dysfunction. Regarding the underlying mechanism, TAX reduced the LPS-induced secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in Caco-2 cell monolayers. In addition, TAX suppressed the phosphorylation of nuclear factor kappa-B (NF-κB), inhibitor protein of NF-κBα (IκBα), and myosin light chain (MLC), and downregulated the expression of myosin light chain kinase (MLCK) in LPS-treated Caco-2 cells. In summary, TAX can maintain TJ proteins by inhibiting the NF-κB/MLCK pathway and pro-inflammatory factor secretion to ameliorate LPS-induced intestinal epithelial barrier dysfunction. Thus, TAX is a promising candidate agent for use in functional food to ameliorate intestinal barrier dysfunction.

Atractylodes oil alleviates diarrhea-predominant irritable bowel syndrome by regulating intestinal inflammation and intestinal barrier via SCF/c-kit and MLCK/MLC2 pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes lancea (Thunb.) DC. is a widely used traditional herb that is well known for treating spleen deficiency and diarrhea. According to traditional Chinese medicine (TCM) theory, diarrhea-predominant irritable bowel syndrome (IBS-D) is caused by cold and dampness, resulting in diarrhea and abdominal pain. Nevertheless, the effect and mechanism of Atractylodes on IBS-D are still unclear. AIM OF THE STUDY: This study was designed to confirm the therapeutic effect of Atractylodes lanceolata oil (AO) in a rat model of IBS-D, and to determine the mechanisms by which AO protects against the disease. MATERIALS AND METHODS: The chemical components in AO were determined using gas chromatography-mass spectrometry (GC-MS). The expression levels of 5-hydroxytryptamine (5-HT), vasoactive intestinal peptide (VIP), and surfactant protein (SP) in serum and colon tissue were measured using enzyme-linked immunosorbent assay (ELISA). Reverse transcription-polymerase chain reaction (RT-PCR), western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF) were used to elucidate the mechanism of action of AO toward inflammation and the intestinal barrier in a rat model of IBS-D. RESULTS: The 15 chemical substances of the highest concentration in AO were identified using GC-MS. AO was effective against IBS-D in the rat model, in terms of increased body weight, diarrhea grade score, levels of interleukin-10 (IL-10), aquaporin 3 (AQP3), and aquaporin 8 (AQP8), and reduced fecal moisture content, levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), 5-HT, VIP, and SP, while also reducing intestinal injury, as observed using hematoxylin-eosin (HE) staining. In addition, the results indicated that AO increased the mRNA and protein expression levels of stem cell factor (SCF) and c-kit and enhanced the levels of zonula occludens-1 (ZO-1) and occludin, as well as decreased the levels of myosin light chain kinase (MLCK) and inhibited the phosphorylation of myosin light chain 2 (p-MLC2). CONCLUSIONS: AO was found to be efficacious in the rat model of IBS-D. AO inhibited the SCF/c-kit pathway, thereby reducing inflammation and protecting against intestinal barrier damage via the MLCK/MLC2 pathway.

Aberrant endoplasmic reticulum stress mediates coronary artery spasm through regulating MLCK/MLC2 pathway.

Coronary artery spasm (CAS) is a pathophysiological phenomenon that may cause myocardial infarction and lead to circulatory collapse and death. Aberrant endoplasmic reticulum (ER) stress causes accumulation of misfolding proteins and has been reported to be involved in a variety of vascular diseases. The present study investigated the role of ER stress in the development of CAS and explored the possible molecular mechanisms. Initially, it was found that ER stress markers were elevated in response to drug-induced vascular smooth muscle cells (VSMCs) contraction. Pharmacologic activation of ER stress using Tunicamycin (Tm) persistently induced CAS and significantly promoted Pituitrin-induced CAS in mice as well as in a collagen gel contraction assay. On the contrary, pharmacologic inhibition of ER stress using 4-phenylacetic acid (4-PBA) completely blunted Pituitrin-induced CAS development in mice. Moreover, during the drug-induced VSMCs contraction, expression of ER stress markers were increased in parallel to those of myosin light chain kinase (MLCK) and phosphor-MLC2 (p-MLC2, at Ser19). After inhibiting MLCK activity by using its specific inhibitor ML-7, the ER stress activator Tm failed to activate the MLCK/MLC2 pathway and could neither trigger CAS in mice nor induce VSMCs contraction in vitro. Our results suggested that aberrant ER stress mediated CAS via regulating the MLCK/MLC2 pathway. ER stress activators might be more robust than the common drugs (Pituitrin or acetylcholine) as to induce vasocontraction and thus may serve as potential therapeutics against chronic bleeding, while its inhibitor might be useful for treatment of severe CAS caused by other medication.

Wogonin inhibits LPS-induced vascular permeability via suppressing MLCK/MLC pathway.

Wogonin, a naturally occurring monoflavonoid extracted from the root of Scutellaria baicalensis Georgi, has been shown to have anti-inflammatory and anti-tumor activities and inhibits oxidant stress-induced vascular permeability. However, the influence of wogonin on vascular hyperpermeability induced by overabounded inflammatory factors often appears in inflammatory diseases and tumor is not well known. In this study, we evaluate the effects of wogonin on LPS induced vascular permeability in human umbilical vein endothelial cells (HUVECs) and investigate the underlying mechanisms. We find that wogonin suppresses the LPS-stimulated hyperactivity and cytoskeleton remodeling of HUVECs, promotes the expression of junctional proteins including VE-Cadherin, Claudin-5 and ZO-1, as well as inhibits the invasion of MDA-MB-231 across EC monolayer. Miles vascular permeability assay proves that wogonin can restrain the extravasated Evans in vivo. The mechanism studies reveal that the expressions of TLR4, p-PLC, p-MLCK and p-MLC are decreased by wogonin without changing the total steady state protein levels of PLC, MLCK and MLC. Moreover, wogonin can also inhibit KCl-activated MLCK/MLC pathway, and further affect vascular permeability. Significantly, compared with wortmannin, the inhibitor of MLCK/MLC pathway, wogonin exhibits similar inhibition effects on the expression of p-MLCK, p-MLC and LPS-induced vascular hyperpermeability. Taken together, wogonin can inhibit LPS-induced vascular permeability by suppressing the MLCK/MLC pathway, suggesting a therapeutic potential for the diseases associated with the development of both inflammatory and tumor.

Involvement of Rho GTPases and their regulators in the pathogenesis of hypertension.

Proper regulation of arterial blood pressure is essential to allow permanent adjustment of nutrient and oxygen supply to organs and tissues according to their need. This is achieved through highly coordinated regulation processes controlling vascular resistance through modulation of arterial smooth muscle contraction, cardiac output, and kidney function. Members of the Rho family of small GTPases, in particular RhoA and Rac1, have been identified as key signaling molecules playing important roles in several different steps of these regulatory processes. Here, we review the current state of knowledge regarding the involvement of Rho GTPase signaling in the control of blood pressure and the pathogenesis of hypertension. We describe how knockout models in mouse, genetic, and pharmacological studies in human have been useful to address this question.