Moxibustion down-regulates colonic epithelial cell apoptosis and repairs tight junctions in rats with Crohn's disease

AIM: To investigate the effects of moxibustion on down-regulation of the colonic epithelial cell apoptosis and repair of the tight junctions in rats with Crohn's disease (CD). METHODS: Sixty male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an herbs-partitioned moxibustion (HPM) group, a mild-warm moxibustion (MWM) group and a salicylazosulphapyridine (SASP) group, with 12 rats in each group. The CD model rats were treated with trinitrobenzene sulphonic acid to induce intestinal inflammation. The rats in the HPM and MWM groups were treated at the Tianshu (ST25) and Qihai (CV6) acupoints once daily for 14 d, and the SASP group was fed SASP twice daily for 14 d. No additional treatment was given to the MC and NC groups. Themicrostructure of the colonic epithelium was observed under a transmission electron microscope, the transepithelial resistance was measured using a shortcircuit current, colonic epithelial cell apoptosis was determined by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labelling assay, and the expression of occludin, claudin-1 and zonula occludens-l (ZO-1) in the colonic epithelial junction was determined by Western blotting and immunofluorescence staining. RESULTS: Compared with the MC group, the microstructure of the colonic epithelial barrier was signifi-cantly improved in rats treated with HPM, MWM or SASP, meanwhile, the current flow was reduced signifi-cantly, with values of 168.20 ± 6.14 vs 99.70 ± 3.13, 99.10 ± 4.28 and 120.30 ± 3.65 mA, respectively (P = 0.001). However, the HPM and MWM groups had higher current flow rates than the SASP group (99.70 ± 3.13, 99.10 ± 4.28 vs 120.30 ± 3.65 mA, P = 0.001). The number of the apoptotic colonic epithelial cells in HPM, MWM and SASP groups was largely reduced (61.5 ± 16.91 vs 15.5 ± 8.89, 14.8 ± 6.27 and 24.7 ± 9.68, respectively (P = 0.001); and the expression of occlu- din, claudin-1 and ZO-1 in the MWM and HPM groups was signifi cantly enhanced (0.48 ± 0.10, 0.64 ± 0.09 vs 0.18 ± 0.05 for occludin, 0.12 ± 0.02, 0.17 ± 0.03 vs 0.05 ± 0.01 for claudin-1, and 0.08 ± 0.01, 0.11 ± 0.01 vs 0.02 ± 0.01 for ZO-1). And in SASP group, the expression of occludin and ZO-1 was also signifi cantly increased (0.27 ± 0.04 vs 0.18 ± 0.05 for occludin and 0.05 ± 0.01 vs 0.02 ± 0.01 for ZO-1), but there was no significant difference for claudin-1. The HPM and MWM groups had higher expression of occludin, claudin-1 and ZO-1 than the SASP group. CONCLUSION: HPM and MWM treatment can down-regulate apoptosis of colonic epithelial cells, repair tight junctions and enhance colonic epithelial barrier function in rats with CD.

CD74 is a survival receptor on colon epithelial cells

AIM: To investigate the expression and function of CD74 in normal murine colon epithelial cells (CEC) and colon carcinoma cells. METHODS: Expression of CD74 mRNA and protein were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and fluorescence-activated cell sorter (FACS). The effect of migration inhibitory factor (MIF) on the survival of normal CEC from C57BL/6, NOD/SCID, and CD74 def icient mice both in vitro and in vivo, and on the CT26 carcinoma cell line was analyzed by (quantitative) qRT-PCR, RTPCR, Western blotting and FACS. RESULTS: CD74 was found to be expressed on normalCEC. Stimulation of CD74 by MIF induced a signaling cascade leading to up-regulation of Bcl-2 expression, resulting in a signif icant increased survival of CEC. CD74 was also expressed on the CT26 colon carcinoma cell line and its stimulation by MIF resulted in enhanced cell survival, up-regulation of Akt phosphorylation and Bcl-2 expression.

Identification of a Native Novel Oncolytic Immunoglobulin on Exfoliated Colon Epithelial Cells: A Bispecific Heterodimeric Chimera of IgA/IgG*

Understanding the nature of cell surface markers on exfoliated colonic cells is a crucial step in establishing criteria for a normally functioning mucosa. We have found that colonic cells isolated from stool samples (SCSR-010 Fecal Cell Isolation Kit, NonInvasive Technologies, Elkridge, MD), preserved at room temperature for up to one week, with viability of >85% and low levels of apoptosis (8% - 10%) exhibit two distinct cell size subpopulations, in the 2.5 μM - 5.0 μM and 5.0 μM - 8.0 μM range. In addition to IgA, about 60% of the cells expressed a novel heterodimeric IgA/IgG immunoglobulin that conferred a broad-spectrum cell mediated cytotoxicity against tumor cells. In a cohort of 58 subjects the exclusive absence of this immunoglobulin in two African-Americans was suggestive of a germline deletion. Serial cultures in stem cell medium retained the expression of this heterodimer. Since a majority of the cystic cells expressed the stem cell markers Lgr5 and Musashi-1 we termed these cells as gastrointestinal progenitor stem cells (GIP-C**). CXCR-4, the cytokine co-receptor for HIV was markedly expressed. These cells also expressed CD20, IgA, IgG, CD45, and COX-2. We assume that they originated from mature columnar epithelium by dedifferentiation. Our observations indicate that we have a robust noninvasive method to study mucosal pathophysiology and a direct method to create a database for applications in regenerative medicine.

Interaction between enteric epithelial cells and Peyer's patch lymphocytes in response to Shigella lipopolysaccharide: Effect on nitric oxide and IL-6 release

AIM： TO investigate the effect of interaction between enteric epithelial cells and lymphocytes of Peyer＇s patch on the release of nitric oxide （NO） and IL-6 in response to Shigella lipopolysaccharide （LPS）. METHODS： Human colonic epithelial cells （Caco-2） were mixed cocultured with lymphocytes of Peyer＇s patch from wild-type （C57 mice） and inducible NO synthase knockout mice, and challenged with Shigella F2a-12 LPS. Release of NO and raiL-6 was measured by Griess colorimetric assay and enzyme-linked immunosorbent assay （ELISA）, respectively. RESULTS： In the absence of LPS challenge, NO was detected in the culture medium of Caco-2 epithelial cells but not in lymphocytes of Peyer＇s patch, and the NO release was further up-regulated in both cocultures with lymphocytes from either the wild-type or iNOS knockout mice, with a significantly higher level observed in the coculture with iNOS knockout lymphocytes. After Shigella F2a-12 LPS challenge for 24-h, NO production was significantly increased in both Caco-2 alone and the coculture with lymphocytes of Peyer＇s patch from the wild-type mice but not from iNOS knockout mice. LPS was found to stimulate the release of mIL-6 from lymphocytes, which was suppressed by coculture with Caco-2 epithelial cells. The LPS-induced mIL-6 production in lymphocytes from iNOS knockout mice was significantly greater than that from the wild-type mice. CONCLUSION： Lymphocytes of Peyer＇s patch maintain a constitutive basal level of NO production from the enteric epithelial cell Caco-2. LPS-induced mIL-6 release from lymphocytes of Peyer＇s patch is suppressed by the cocultured epithelial cells. While no changes are detectable in NO production in lymphocytes from both wild-type and iNOS knockout mice before and after LPS challenge, NO from lymphocytes appears to play an inhibitory role in epithelial NO release and their own mIL-6 release in response to LPS.

A 75 kDa glycoprotein isolated from Cudrania tricuspidata Bureau induces colonic epithelial proliferation and ameliorates mouse colitis induced by dextran sulfate sodium

Cudrania tricuspidata Bureau(CTB),a species of the Moraceae plant,has been used as a bruise recovery treatment.This study aimed to determine whether the 75 kDa phytoglycoprotein extracted from CTB has a regulatory effect on the proliferation of human colon epithelial cells and the pathological process of inflammatory bowel disease(IBD).We found that CTB glycoprotein significantly induces the proliferation of human colon epithelial HT-29 cells by activating protein kinase C.CTB glycoprotein stimulated the phosphorylation of c-Jun N-terminal kinase and transcription factor nuclear factor-κB,which are responsible for the expression of cell-cycle-related proteins(CDK2,CDK4,cyclin D1 and cyclin E)during its promotion of cell proliferation.Experimental colitis was induced in mice by adding dextran sulfate sodium to their drinking water at a concentration of 4%(W/V)for seven days.We found that CTB glycoprotein ameliorates the pathological process of IBD and lowers the disease activity index score,which was composed of body weight change,diarrhea,and hematochezia in ICR mice treated with dextran sulfate sodium.Hence,we suggest that CTB glycoprotein has the ability to prevent IBD by promoting cell proliferation signaling events via the activation of PKC,JNK and NF-κB in colon epithelial cells.

Nicotine protects against ulcerative colitis through regulating microRNA-124 and STAT3

OBJECTIVE Although it is generally believed that nicotine accounts for the beneficial effect of smoking on ulcerative colitis,the underlying mechanisms remain not well-understood.Our previous finding that nicotine inhibits inflammatory responses through inducing miRNA-124 prompted us to ask whether the miRNA is involved in the protective action of nicotine on UC.METHODS Mi R-124 expression in colon tissues and cells was determined by q-PCR and in situ hybridization.The effect of miR-124 on protective role of nicotine in ulcerative colitis was evaluated in DSS-treated mice and IL-6-treated Caco-2 colon epithelial cells.Expression of p-STAT3/STAT3 was detected by immunohistochemistry and Western blot analysis.RESULTS miR-124 expression is upregulated in colon tissues from patients and DSS-induced colitis.Nicotine treatment further elevated miR-124 level in colon tissues of the mice,in infiltrated lymphocytes and epithelial cells,and augmented miR-124 expression in lymphocytes isolated from human ulcerative colon tissues.Administration of nicotine also reduced weight loss,improved DAI and decreased HE score in DSS-induced colitis.Moreover,knockdown of miR-124 in vivo significantly diminished the beneficial effect of nicotine,and in vitro on IL-6-treated Caco-2 colon epithelial cells.Further analysis indicated that nicotine inhibited STAT3 activation in vivo and in IL-6-treated Caco-2 colon epithelial cells and Jurkat human T lymphocytes,in whichmiR-124 knockdown led to increased activation of STAT3.CONCLUSION These data indicated that nicotine exerts its protective action in UC through inducing miR-124 and its effect on STAT3,suggesting that the miR-124/STAT3 system is a potential target for the therapeutic intervention of UC.

Germline Deletion of the Expression of a Human Bispecific Mucosal Immunoglobulin: Genetic Predisposition to Cancer and Communicable Diseases Predominantly among African-Americans

In a previous report we had reported on the discovery of a novel bispecific immunoglobulin expressed by colonic epithelial cells as they transform into immunomimetic cells during exfoliation (Albaugh et al. (2020) Open Journal of Preventive Medicine, 10, 126-150). Colonic cells isolated from 0.5 gm aliquots of fresh stools (SCSR-10, Fecal Cell Isolation Kit, NonInvasive Technologies, Elkridge, MD) preserved at room temperature for up to one week, with viability of >85% were used to determine the number of cells expressing this novel bispecific immunoglobulin. Over the course of this period (18 years) we recognized that these cells opened the opportunity to investigate the expression of cell membrane biomarkers. As the applications grew, we introduced a new terminology, termed COPROCYTOBIOLOGY*. In this study, we surveyed a cohort of 58 free-living adults for the expression of the newly discovered bi-specific chimeric antibody. Almost all of the subjects showed a strong signal during flow-cytometric evaluation of their stool samples;averaging around 65%. However, two subjects exhibited a total loss of this signal and both these individuals were of African-American lineage (one male and one female). These cells upon culturing in vitro remained defective in contrast to the rest of the group where their progeny continued to generate the antibody. We propose that this signals the existence of a germ-line deletion of the gene for which a novel test (MEDISHIELD†) is suggested. This syndrome may be associated with a lack of response to prophylactic vaccines involving m-RNA.

Experimental Study on the Molecular Mechanism of Anthraquinone Cathartics in Inducing Melanosis Coli

Objective：To explore the significance of colonic epithelial cell apoptosis and tumor necrosis factorα（TNF-α）changing in pathogenesis of melanosis coli（MC）in guinea pig and the molecular mechanism of rhubarb（Rhu）in inducing the disease,by means of using different dosages of Rhu to induce the disease. Methods：One hundred and forty-four male guinea pigs,clean grade,were randomized according to their body weight into 5 groups,the untreated normal group and the 4 Rhu groups treated,respectively,with different doses of Rhu,3 g/kg·d for low dose（Rhu-I）group,6 g/kg·d for moderate dose（Rhu-m）group,12 g/kg·d for high dose（Rhu-h）group and 24 g/kg·d for super-high dose（Rhu-s）group via gastric infusion.All animals were sacrificed 60 days later,their viscera were taken for observing the pathologic and morphologic changes with HE, melanin and melatonin staining,and the apoptosis of colonic epithelial cells was detected with TUNEL stain and transmission electric microscopy.In addition,the levels of TNF-αin serum and colonic tissue were measured using ELISA and RT-PCR.Results：The pathological changes of MC could be found by naked eye in all Rhu groups,especially apparent at caecum and proximal end of colon,but did not found in gallbladder,jejunum and ileum.In normal guinea pigs,the colonic membrane was pink in color with no apparent pigment deposition. Membranous color deepened in the Rhu groups depending on the dosage of Rhu used.MC scoring showed the highest scores revealed in the Rhu-s group（6.00±0.00）,which was significantly different to those in the Rhu-I （3.86±0.69）,Rhu-m（4.43±0.79）and Rhu-h groups（4.88±0.35,all P0.05）.Levels of cell apoptosis in colon and TNF-αin serum in all Rhu groups were higher than those in the normal group（P0.01）,but showed no significant difference among the Rhu groups（P0.05）.Moreover,a positive correlation was found in the degree of induced MC with apoptosis rate and TNF-αlevel.Conclusions：Rhu（anthraquinone purgatives）had apparent effect on inducing MC;its molecular mechanism is maybe to destroy intestinal mucosal barrier and advance proinflammatory factor TNF-αreleasing,which leads to colonic epithelial cells apoptosis,and finally induce the change of MC due to the deposition of brown pigments,i.e.the macrophage phagocytized apoptotic body,on the colonic membrane.