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Gut microbial preparations are widely used in treating intestinal diseases but show mixed success. In this study, we found that the therapeutic efficacy of A. muciniphila for dextran sodium sulfate (DSS)-induced colitis as well as intestinal radiation toxicity was ~50%, and mice experiencing a positive prognosis harbored a high frequency of A. muciniphila in the gastrointestinal (GI) tract. Stable GI colonization of A. muciniphila elicited more profound shifts in the gut microbial community structure of hosts. Coexisting with A. muciniphila facilitated proliferation and reprogrammed the gene expression profile of Lactobacillus murinus, a classic probiotic that overtly responded to A. muciniphila addition in a time-dependent manner. Then, a magnetic-drove, mannose-loaded nanophase material was designed and linked to the surface of A. muciniphila. The modified A. muciniphila exhibited enhancements in inflammation targeting and intestinal colonization under an external magnetic field, elevating the positive-response rate and therapeutic efficacy against intestinal diseases. However, the unlinked cocktail containing A. muciniphila and the delivery system only induced negligible improvement of therapeutic efficacy. Importantly, heat-inactivated A. muciniphila lost therapeutic effects on DSS-induced colitis and was even retained in the GI tract for a long time. Further investigations revealed that the modified A. muciniphila was able to drive M2 macrophage polarization by upregulating the protein level of IL-4 at inflammatory loci. Together, our findings demonstrate that stable colonization of live A. muciniphila at lesion sites is essential for its anti-inflammatory function.

Social hierarchy governs the physiological and biochemical behaviors of animals. Intestinal radiation injuries are common complications connected with radiotherapy. However, it remains unclear whether social hierarchy impacts the development of radiation-induced intestinal toxicity. Dominant mice exhibited more serious intestinal toxicity following total abdominal irradiation compared with their subordinate counterparts, as judged by higher inflammatory status and lower epithelial integrity. Radiation-elicited changes in gut microbiota varied between dominant and subordinate mice, being more overt in mice of higher status. Deletion of gut microbes by using an antibiotic cocktail or restructuring of the gut microecology of dominant mice by using fecal microbiome from their subordinate companions erased the difference in radiogenic intestinal injuries. Lactobacillus murinus and Akkermansia muciniphila were both found to be potential probiotics for use against radiation toxicity in mouse models without social hierarchy. However, only Akkermansia muciniphila showed stable colonization in the digestive tracts of dominant mice, and significantly mitigated their intestinal radiation injuries. Our findings demonstrate that social hierarchy impacts the development of radiation-induced intestinal injuries, in a manner dependent on gut microbiota. The results also suggest that the gut microhabitats of hosts determine the colonization and efficacy of foreign probiotics. Thus, screening suitable microbial preparations based on the gut microecology of patients might be necessary in clinical application.

Radiation-induced cardiopulmonary injuries are the most common and intractable side effects that are entwined with radiotherapy for thorax cancers. However, the therapeutic options for such complications have yielded disappointing results in clinical applications. Here, we reported that gut microbiota-derived l-Histidine and its secondary metabolite imidazole propionate (ImP) fought against radiation-induced cardiopulmonary injury in an entiric flora-dependent manner in mouse models. Local chest irradiation decreased the level of l-Histidine in fecal pellets, which was increased following fecal microbiota transplantation. l-Histidine replenishment via an oral route retarded the pathological process of lung and heart tissues and improved lung respiratory and heart systolic function following radiation exposure. l-Histidine preserved the gut bacterial taxonomic proportions shifted by total chest irradiation but failed to perform radioprotection in gut microbiota-deleted mice. ImP, the downstream metabolite of l-Histidine, accumulated in peripheral blood and lung tissues following l-Histidine replenishment and protected against radiation-induced lung and heart toxicity. Orally gavaged ImP could not enter into the circulatory system in mice through an antibiotic cocktail treatment. Importantly, ImP inhibited pyroptosis to nudge lung cell proliferation after radiation challenge. Together, our findings pave a novel method of protection against cardiopulmonary complications intertwined with radiotherapy in pre-clinical settings and underpin the idea that gut microbiota-produced l-Histidine and ImP are promising radioprotective agents.

Safe and effective regimens are still needed given the risk of radiation toxicity from iatrogenic irradiation. The gut microbiota plays an important role in radiation damage. Diet has emerged as a key determinant of the intestinal microbiome signature and function. In this report, we investigated whether a 30% caloric restriction (CR) diet may ameliorate radiation enteritis and hematopoietic toxicity. Experimental mice were either fed ad libitum (AL) or subjected to CR preconditioning for 10 days and then exposed to total body irradiation (TBI) or total abdominal irradiation (TAI). Gross examinations showed that short-term CR pretreatment restored hematogenic organs and improved the intestinal architecture in both male and female mice. Intriguingly, CR preconditioning mitigated radiation-induced systemic and enteric inflammation in female mice, while gut barrier function improved in irradiated males. 16S rRNA high-throughput sequencing showed that the frequency of pro-inflammatory microbes, including Helicobacter and Desulfovibrionaceae, was reduced in female mice after 10 days of CR preconditioning, while an enrichment of short-chain fatty acid (SCFA)-producing bacteria, such as Faecalibaculum, Clostridiales, and Lactobacillus, was observed in males. Using fecal microbiota transplantation (FMT) or antibiotic administration to alter the gut microbiota counteracted the short-term CR-elicited radiation tolerance of both male and female mice, further indicating that the radioprotection of a 30% CR diet depends on altering the gut microbiota. Together, our findings provide new insights into CR in clinical applications and indicate that a short-term CR diet prior to radiation modulates sex-specific gut microbiota configurations, protecting male and female mice against the side effects caused by radiation challenge.

Radioresistance is a major challenge in lung cancer radiotherapy (RT), and consequently, new radiosensitizers are urgently needed. MicroRNAs (miRNAs) have been demonstrated to participate in many important cellular processes including radiosensitization. MiR-365 is dysregulated in non-small cell lung cancer (NSCLC) and is able to restrain the development of NSCLC. However, the relationship between miR-365 and radiosensitivities of NSCLC cells remains largely unknown. Here we reveal that overexpression of miR-365 is able to enhance the radiosensitivity of NSCLC cells through targeting CDC25A. We found that the expression level of miR-365 was positively correlated with the radiosensitivity of NSCLC cell lines. Furthermore, our results showed that overexpression of miR-365 could sensitize A549 cells to the irradiation. However, knockdown of miR-365 in H460 cells could act the converse manner. Mechanically, miR-365 was able to directly target 3′UTR of cell division cycle 25A (CDC25A) mRNA and reduce the expression of CDC25A at the levels of mRNA and protein. And we confirmed that miR-365 could increase the radiosensitivity of NSCLC cells by targeting CDC25A using in vitro and in vivo assays. Taken together, restoration of miR-365 expression enhances the radiosensitivity of NSCLC cells by suppressing CDC25A, and miR-365 could be used as a radiosensitizer for NSCLC therapy.

Introduction Radioresistance is a major challenge in lung cancer radiotherapy, and new radiosensitizers are urgently needed. Estrogen receptor β (ERβ) is involved in the progression of non-small cell lung cancer (NSCLC), however, the role of ERβ in the response to radiotherapy in lung cancer remains elusive. In the present study, we investigated the mechanism underlying ERβ-mediated transcriptional activation and radioresistance of NSCLC cells. Methods Quantitative real-time PCR, western blot and immunohistochemistry were used to detect the expression of CLPTM1L, ERβ and other target genes. The mechanism of CLPTM1L in modulation of radiosensitivity was investigated by chromatin immunoprecipitation assay, luciferase reporter gene assay, immunofluorescence staining, confocal microscopy, coimmunoprecipitation and GST pull-down assays. The functional role of CLPTM1L was detected by function assays in vitro and in vivo. Results CLPTM1L expression was negatively correlated with the radiosensitivity of NSCLC cell lines, and irradiation upregulated CLPTM1L in radioresistant (A549) but not in radiosensitive (H460) NSCLC cells. Meanwhile, IR induced the translocation of CLPTM1L from the cytoplasm into the nucleus in NSCLC cells. Moreover, CLPTM1L induced radioresistance in NSCLC cells. iTRAQ-based analysis and cDNA microarray identified irradiation-related genes commonly targeted by CLPTM1L and ERβ, and CLPTM1L upregulated ERβ-induced genes CDC25A, c-Jun, and BCL2. Mechanistically, CLPTM1L coactivated ERβ by directly interacting with ERβ through the LXXLL NR (nuclear receptor)-binding motif. Functionally, ERβ silencing was sufficient to block CLPTM1L-enhanced radioresistance of NSCLC cells in vitro. CLPTM1L shRNA treatment in combination with irradiation significantly inhibited cancer cell growth in NSCLC xenograft tumors in vivo. Conclusions The present results indicate that CLPTM1L acts as a critical coactivator of ERβ to promote the transcription of its target genes and induce radioresistance of NSCLC cells, suggesting a new target for radiosensitization in NSCLC therapy.

Radiotherapy is often employed to treat abdominal and pelvic malignancies, but is frequently accompanied by diverse acute and chronic local injuries. It was previously unknown whether abdominal and pelvic radiotherapy impairs distant cognitive dysfunction. In the present study, we demonstrated that total abdominal irradiation (TAI) exposure caused cognitive deficits in mouse models. Mechanically, microarray assay analysis revealed that TAI elevated the expression level of miR-34a-5p in small intestine tissues and peripheral blood (PD), which targeted the 3′UTR of Brain-derived neurotrophic factor ( Bdnf ) mRNA in hippocampus to mediate cognitive dysfunction. Tail intravenous injection of miR-34a-5p antagomir immediately after TAI exposure rescued TAI-mediated cognitive impairment via blocking the up-regulation of miR-34a-5p in PD, resulting in restoring the Bdnf expression in the hippocampus. More importantly, high throughput sequencing validated that the gut bacterial composition of mice was shifted after TAI exposure, which was retained by miR-34a-5p antagomir injection. Thus, our findings provide new insights into pathogenic mechanism underlying abdominal and pelvic radiotherapy-mediated distant cognitive impairment.

Radiotherapy significantly improves the therapeutic outcomes and survival of breast cancer patients. However, the acquired resistance to this therapeutic modality is a major clinical challenge. Here we show that ionizing irradiation (IR)-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3) at the Tyr705 residue and the induction of reactive oxygen species (ROS) in wild-type and radioresistant MDA-MB-231 and MDA-MB-468 triple-negative breast cancer (TNBC) cell lines. Comparing with radiosensitive parental TNBC cells, significantly low levels of ROS and higher protein levels of phospho-STAT3 and Bcl-2 were observed in TNBC cells with acquired radioresistance. Moreover, knockdown of STAT3 by shRNA sensitized the TNBC cells to IR. Niclosamide, a potent inhibitor of STAT3, overcame the radioresistance in TNBC cells via inhibition of STAT3 and Bcl-2 and induction of ROS. In combination with radiation, niclosamide treatment resulted in significant increase of ROS generation and induction of apoptosis in parental and radioresistant TNBC cells in vitro and TNBC xenograft tumors in vivo. These findings demonstrate that activation of STAT3 and Bcl-2 and reduction of ROS contribute to the development of radioresistance in TNBC, and niclosamide acts as a potent radiosensitizer via inhibiting STAT3 and Bcl-2 and increasing ROS generation in TNBC cells and xenograft tumors. Our findings suggest that niclosamide in combination with irradiation may offer an effective alternative approach for restoring the sensitivity of radioresistant TNBC cells to IR for improved therapeutic efficacy and outcomes.

Irradiation exposure positive correlates with tumor formation, such as breast cancer and lung cancer. However, whether low dose irradiation induces hepatocarcinogenesis and the underlying mechanism remain poorly defined. In the present study, we reported that low dose irradiation facilitated the proliferation of hepatocyte through up-regulating HULC in vitro and in vivo. Low dose irradiation exposure elevated HULC expression level in hepatocyte. Deletion of heightened HULC erased the cells growth accelerated following low dose irradiation exposure. CDKN1, the neighbor gene of HULC, was down-regulated by overexpression of HULC following low dose irradiation exposure via complementary base pairing, resulting in promoting cell cycle process. Thus, our findings provide new insights into the mechanism of low dose irradiation-induced hepatocarcinogenesis through HULC/CDKN1 signaling, and shed light on the potential risk of low dose irradiation for the development of hepatocellular carcinoma in pre-clinical settings.

B7-H3 is a glycoprotein overexpressed in cancer, but its functional contribution in this setting remains poorly understood. In the present study, we identified that the overexpression of B7-H3 in lung cancer resulted in aberrant lipid metabolism via SREBP-1/FASN signaling pathway. Immunohistochemical analysis of tissue microarrays revealed that approximately 80.4% (37/46) of lung cancer tissues were positive for B7-H3 accompanying poor prognosis. Notably, Oil red O staining and total triglyceride assay exhibited that down-regulation of B7-H3 decreased lipid synthesis in lung cancer A549 and H446 cell lines. Mechanistic investigations showed that B7-H3 modulated the expression of FASN, a fatty acid synthase, specifically. Furthermore, deletion of B7-H3 down-regulated the mRNA and protein levels of SREBP-1, a transcription factor governing the expression of FASN. Finally, correlation analysis between expression levels of B7-H3 and FASN exhibited a positive correlation in clinical lung cancer tissues. Overall, we conclude that B7-H3 hijacks SREBP-1/FASN signaling mediating abnormal lipid metabolism in lung cancer. Our finding provides new insights into the function and mechanism of B7-H3 in the development of lung cancer.

Although radiation therapy is a cornerstone of modern management of malignancies, various side effects are inevitably linked to abdominal and pelvic cancer after radiotherapy. Radiation-mediated gastrointestinal (GI) toxicity impairs the life quality of cancer survivors and even shortens their lifespan. Hydrogen has been shown to protect against tissue injuries caused by oxidative stress and excessive inflammation, but its effect on radiation-induced intestinal injury was previously unknown. In the present study, we found that oral gavage with hydrogen-water increased the survival rate and body weight of mice exposed to total abdominal irradiation (TAI); oral gavage with hydrogen-water was also associated with an improvement in GI tract function and the epithelial integrity of the small intestine. Mechanistically, microarray analysis revealed that hydrogen-water administration upregulated miR-1968-5p levels, thus resulting in parallel downregulation of MyD88 expression in the small intestine after TAI exposure. Additionally, high-throughput sequencing showed that hydrogen-water oral gavage resulted in retention of the TAI-shifted intestinal bacterial composition in mice. Collectively, our findings suggested that hydrogen-water might be used as a potential therapeutic to alleviate intestinal injury induced by radiotherapy for abdominal and pelvic cancer in preclinical settings.

Phosphate homeostasis is critical for many physiological functions. Up to 85% of phosphate is stored in bone and teeth. The remaining 15% is distributed in cells. Phosphate absorption across the brush-border membrane (BBM) of enterocytes occurs mainly via a sodium-dependent pathway, which is mediated by type IIb sodium-phosphate cotransporters (NaPi-IIb). Patients of inflammatory bowel diseases (IBDs) suffer not only from diarrhea and nutrient malabsorption but also from bone loss. About 31-59% of patients with IBD develop bone disorders. Since the intestine is a primary location for dietary phosphate absorption, it is logical to postulate that there is an inverse relationship between gastrointestinal disorders and phosphate transport, which, in turn, contributes to bone disorders observed in patients with IBD. Phosphate absorption and NaPi-IIb expression was studied with BBM vesicles isolated from trinitrobenzene sulphonic acid (TNBS) animals as well as in Caco-2 cells. The mechanism of TNF-alpha downregulation of NaPi-IIb expression was investigated by luciferase assay, gel mobility shift assay (GMSA), and coimmunoprecipitation. Intestinal phosphate absorption mediated by NaPi-IIb was reduced both in TNBS colitis and in TNF-alpha-treated cells. Transient transfection indicated that TNF-alpha inhibits NaPi-IIb expression by reducing NaPi-IIb basal promoter activity. GMSAs identified NF1 protein as an important factor in TNF-alpha-mediated NaPi-IIb downregulation. Signaling transduction study and coimmunoprecipitation suggested that TNF-alpha interacts with EGF receptor to activate ERK1/2 pathway. Intestinal phosphate absorption mediated by NaPi-IIb protein is reduced in colitis. This inhibition is mediated by the proinflammatory cytokine TNF-alpha through a novel molecular mechanism involving TNF-alpha/EGF receptor interaction.

Our previous studies have identified a minimal Sp1-driven promoter region (nt −36/+116) directing NHE2 expression in mouse renal epithelial cells. However, this minimal promoter region was not sufficient to support active transcription of NHE2 gene in the intestinal epithelial cells, suggesting the need for additional upstream regulatory elements. In the present study, we used nontransformed rat intestinal epithelial (RIE) cells as a model to identify the minimal promoter region and transcription factors necessary for the basal transcription of rat NHE2 gene in the intestinal epithelial cells. We identified a region within the rat NHE2 gene promoter located within nt −67/−43 upstream of transcription initiation site as indispensable for the promoter function in intestinal epithelial cells. Mutations at nt −56/−51 not only abolished the DNA-protein interaction in this region, but also completely abolished NHE2 gene promoter activity in RIE cells. Supershift assays revealed that Sp1 and Sp3 interact with this promoter region, but, contrary to the minimal promoter indispensable for renal expression of NHE2, both transcription factors expressed individually in Drosophila SL2 cells activated rat NHE2 gene promoter. Moreover, Sp1 was a weaker transactivator and when coexpressed in SL2 cells it reduced Sp3-mediated NHE2 basal promoter activity. Furthermore, DNase I footprinting confirmed that nt −58/−51 is protected by nuclear protein from RIE cells. We conclude that the mechanism of basal control of rat NHE2 gene promoter activity is different in the renal and intestinal epithelium, with Sp3 being the major transcriptional activator of NHE2 gene transcription in the intestinal epithelial cells.

Extracts from the Manchurian walnut have the antibacterial, anti-inflammatory, and anticancer properties. In this study, different extracts were obtained from Manchurian walnut peels, flowers, and leaves by the Soxhlet extraction method, and used to study the antitumor activity. MTT assay shows that the ethyl acetate phase of the peels inhibits cell proliferation of colon cancer cell SW480 with an IC50 at 56 μg/mL. Hoechst 33258 staining illustrates that the ethyl acetate phase of the peels induces cell apoptosis of SW480 cells. These results indicate that the ethyl acetate of Manchurian walnut peels inhibits cell proliferation and promotes cell apoptosis of cancer cells.

To understand the mechanism(s) underlying stress responses and discover new stress-tolerance genes in rice ( Oryza sativa L.), expression profiles were obtained for leaf and panicle tissues at seedling, booting and heading stages of indica cultivar Pei’ai 64S plants under cold, drought or heat stresses using the GeneChip Rice Genome Array (Affymetrix) representing 51, 279 transcripts from japonica and indica rice. A large number of genes highly up regulated or down regulated were identified under the stresses. OsXET9 ( O. sativa L. xyloglucan endotransglucosylase 9, GenBank accession: NM_001060322) was highly induced in leaf and panicle at all the developmental stages, in response to all stresses, especially in seedling and booting stage under cold stress. Real-time quantitative PCR analysis showed that the result was consensus with GeneChip Rice Genome Array, suggesting that OsXET9 is a multiple stress responsive gene in rice. In order to study its function in stress tolerance, we cloned the cDNA of the gene through amplification by PCR. Sequence analysis showed that the cDNA encodes a protein of 284 amino acid residues with M.W. ≈ 31kD and pI ≈ 5.2. The gene encodes a protein with several conserved domains. Comparison of protein sequences indicates that OsXET9 is closely related to the xyloglucan endotransglycosylase. Analysis of the putative promoter region for candidate cis-regulatory elements using Plant CARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) identified some cis-elements related to stress responses. Based on these analyses and results obtained, we propose that OsXET9 is a novel candidate gene involved in stress tolerance in rice in addition to the function in metabolism of cell wall. Keywords : Rice, abiotic stress, microarray, xyloglucan endotransglucosylase.

Modern lifestyles, such as shift work, nocturnal social activities, and jet lag, disturb the circadian rhythm. The interaction between mammals and the co-evolved intestinal microbiota modulates host physiopathological processes. Radiotherapy is a cornerstone of modern management of malignancies; however, it was previously unknown whether circadian rhythm disorder impairs prognosis after radiotherapy. To investigate the effect of circadian rhythm on radiotherapy, C57BL/6 mice were housed in different dark/light cycles, and their intestinal bacterial compositions were compared using high throughput sequencing. The survival rate, body weight, and food intake of mice in diverse cohorts were measured following irradiation exposure. Finally, the enteric bacterial composition of irradiated mice that experienced different dark/light cycles was assessed using 16S RNA sequencing. Intriguingly, mice housed in aberrant light cycles harbored a reduction of observed intestinal bacterial species and shifts of gut bacterial composition compared with those of the mice kept under 12 h dark/12 h light cycles, resulting in a decrease of host radioresistance. Moreover, the alteration of enteric bacterial composition of mice in different groups was dissimilar. Our findings provide novel insights into the effects of biological clocks on the gut bacterial composition, and underpin that the circadian rhythm influences the prognosis of patients after radiotherapy in a preclinical setting.

Abiotic stresses constitute a serious threat to agricultural production, which often develops into major crop production reducing factors around the world. Molecular biology technology has, however, emerged as a promising vehicle improving crop tolerance. A cold-, drought- and heat-inducible gene designated Oryza sativa L. abscisic acid stress-ripening ( OsASR ) gene, GenBank accession: AK318549.1 was identified in rice Pei’ai64s ( O. sativa L. ssp. Indica cv.) using the GeneChip rice genome array (Affymetrix) representing 51, 279 transcripts from two rice subspecies japonica and indica . The expression profile of OsASR obtained by the microarray analysis was confirmed by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of the gene. The two sets of data matched very well, suggesting that OsASR is a multiple stresses responsive gene in rice. Based on the sequence, PCR primers were designed. The cDNA with the whole open reading frame (ORF) was amplified by PCR and cloned. Sequence analysis showed that the cDNA encodes a protein of 284 amino acid residues with M.W. ≈ 11.7 kD and pI ≈ 10.4. The gene encodes a protein with several conserved domains. Comparison of protein sequences indicates that OsASR encodes a putative abscisic acid stress-ripening protein. Analysis of the putative promoter region for candidate cis-regulatory elements using PlantCARE software identified seven kinds of cis-elements related to stress responses. Based on the aforementioned analyses and results obtained, we propose that OsASR is a novel candidate gene involved in stress tolerance in rice. Keywords: Rice, microarray, abiotic stress, reverse transcription polymerase chain reaction (RT-PCR), abscisic acid stress ripening

To understand mechanisms underlying stress responses and discover new stress-tolerance genes in rice (Oryza sativa L.), we analyzed a global genome expression profiling of the indica cultivar Pei’ai 64S subjected to cold, drought, or heat stresses. Expression profiles were obtained for leaf and panicle tissues at seedling, booting and heading stages from plants under no stress, or cold, drought or heat stresses using the GeneChip Rice Genome Array (Affymetrix) representing 51 279 transcripts from japonica and indica rice. A large number of genes highly up- regulated or down-regulated were identified under the stresses. One of these genes, OsMyb1R, was highly induced in leaf and panicle at the heading and flowering stages, in response to all stresses. The expression profile of OsMyb1R obtained by the microarray analysis was confirmed by quantitative real-time reverse transcriptaseplolymerase chain reaction (RT-PCR) analysis of the gene. The two sets of data matched very well, suggesting that OsMyb1R is a multiple stresses responsive gene in rice. In order to study its function in stress tolerance, we cloned the cDNA of the gene through amplification by RT-PCR. Sequence analysis showed that the cDNA encodes a protein of 489 amino acid residues with molecular weight (M.W.) ≈ 56 kD and pI ≈ 6. Bioinformatics analysis revealed that the protein contains a conservative domain of the MYB family gene. Analysis of the putative promoter region for candidate cis-regulatory elements identified 5 matches to cis-elements related to stress responses. Based on the earlier mentioned analysis and results obtained, we propose that OsMyb1R is a novel candidate gene involved in stress tolerance in rice.Key words: Oryza sativa L., stress, microarray, real-time PCR, MYB, bioinformatics.

NHE8 transporter is a member of the sodium/hydrogen exchanger (NHE) family. This transporter protein is expressed at the apical membrane of epithelial cells of kidney and intestine and contributes to vectorial Na+transport in both tissues. Although NaCl absorption has been shown to be reduced in diarrhea associated with colitis and enteritis, little is known about the role of Na+/H+exchange and the involvement of NHE isoforms in the pathogenesis of inflammatory disorders and the mechanism of inflammation-associated diarrhea. This study investigated the role of NHE8 in the setting of inflammatory states. Jejunal mucosa was harvested from trinitrobenzene sulfonic acid (TNBS) colitis rats or lipopolysaccharide (LPS) rats for RNA extraction and brush-border membrane protein purification. The human NHE8 gene promoter was cloned from human genomic DNA and characterized in Caco-2 cells. The promoter was further used to study the mechanisms of TNF-α-mediated NHE8 expression downregulation in Caco-2 cells. Results from Western blot and real-time PCR indicated that NHE8 protein and mRNA were significantly reduced in TNBS rats and LPS rats. In Caco-2 cells, TNF-α produces similar reduction levels in the endogenous NHE8 mRNA expression observed in our in vivo studies. The downregulation of NHE8 expression mediated by TNF-α could be blocked by transcription inhibitor actinomycin D, suggesting the involvement of transcriptional regulation. Further studies indicated that the human NHE8 gene transcription could be activated by Sp3 transcriptional factor, and TNF-α inhibits human NHE8 expression by reducing Sp3 interaction at the minimal promoter region of the human NHE8 gene. In conclusion, our studies suggest that TNF-α decreases NHE8 expression in inflammation induced by TNBS and LPS, which may contribute to the diarrhea associated with inflammation.

NHE8 is a newly identified NHE isoform expressed in rat intestine. To date, the kinetic characteristics and the intestinal segmental distribution of this NHE isoform have not been studied. This current work was performed to determine the gene expression pattern of the NHE8 transporter along the gastrointestinal tract, as well as its affinity for Na(+), H(+), and sensitivity to known NHE inhibitors HOE694 and S3226. NHE8 was differentially expressed along the GI tract. Higher NHE8 expression was seen in stomach, duodenum, and ascending colon in human, while higher NHE8 expression was seen in jejunum, ileum and colon in adult mouse. Moreover, the expression level of NHE8 is much higher in the stomach and jejunum in young mice compared with adult mice. To evaluate the functional characterictics of NHE8, the pH indicator SNARF-4 was used to monitor the rate of intra-cellular pH (pH(i)) recovery after an NH(4)Cl induced acid load in NHE8 cDNA transfected PS120 cells. The NHE8 cDNA transfected cells exhibited a sodium-dependent proton exchanger activity having a Km for pH(i) of approximately pH 6.5, and a Km for sodium of approximately 23 mM. Low concentration of HOE694 (1 microM) had no effect on NHE8 activity, while high concentration (10 microM) significantly reduced NHE8 activity. In the presence of 80 microM S3226, the NHE8 activity was also inhibited significantly. In conclusion, our work suggests that NHE8 is expressed along the gastrointestinal tract and NHE8 is a functional Na(+)/H(+) exchanger with kinetic characteristics that differ from other apically expressed NHE isoforms.