Targeting mitochondrial transcription factor A sensitizes pancreatic cancer cell to gemcitabine.

BACKGROUND: The survival of pancreatic cancer cells, particularly cancer stem cells which are responsible for tumor relapse, depends on mitochondrial function. Mitochondrial transcription factor A (TFAM) is critical for the regulation of mitochondrial DNA and thus mitochondrial function. However, the possible involvement of TFAM in pancreatic cancer is unknown. METHODS: Human samples were obtained from pancreatic cancers and their adjacent tissues; human pancreatic cell lines were cultured in RPMI1640 medium. TFAM expressions in pancreatic tissues and cultured cells were determined using immunohistochemistry, ELISA, and reverse transcription polymerase chain reaction (RT-PCR). The effect of TFAM on cell growth, migration, colony formation and apoptosis were evaluated. Mitochondrial biogenesis in pancreatic cancer and normal cells were examined. RESULTS: The majority of pancreatic cancer tissues exhibited higher TFAM expression compared to the adjacent counterparts. Consistently, TFAM mRNA and protein levels were higher in pancreatic cancer cell lines than in immortalized normal pancreatic epithelial cells. There was no difference on TFAM level between gemcitabine-sensitive and resistant pancreatic cancer cells. Functional analysis demonstrated that TFAM overexpression activated pancreatic normal and tumor cells whereas TFAM inhibition effectively inhibited the growth of pancreatic cancer cells. TFAM inhibition enhanced gemcitabine's cytotoxicity and suppressed growth, anchorage-independent colony formation and survival of gemcitabine-resistant pancreatic cancer cells. Mechanistic studies showed that TFAM inhibition resulted in remarkable mitochondrial dysfunction and energy crisis followed by oxidative stress. The basal mitochondrial biogenesis level correlated well with TFAM level in pancreatic cancer cells. CONCLUSIONS: TFAM played essential roles in pancreatic cancer via regulating mitochondrial functions which highlighted the therapeutic value of inhibiting TFAM to overcome gemcitabine resistance.

The impact of acute stress disorder on gallbladder interstitial cells of Cajal.

Physical and psychological stress exerts a substantial effect on gastrointestinal motility disorders, where trauma enhances symptoms of digestive dysfunction. Interstitial cells of Cajal (ICCs) act as pacemakers for gastrointestinal motility regulation and are likely important in stress-associated gastrointestinal motility disorders. This study explored the mechanisms underlying gallbladder ICCs function under acute stress conditions using a rabbit chest puncture and cholecystectomy model. The stem cell factor (SCF)/c-kit pathway is essential for the development of ICCs, and gene expression was investigated to identify stress-induced transcriptional alterations. Immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling assays were used to determine ICCs apoptosis, whereas western blot analysis and reverse-transcription polymerase chain reaction were used to detect changes in the SCF/c-kit signaling pathway. These methods revealed a reduction in ICCs via apoptosis following stress, and ICCs increased over time after stressor removal. Therefore, this study demonstrates the impact of stress on ICCs development and survival and further confirms the link between stress and gastrointestinal motility.

Clinical characteristics of patients with 2019 coronavirus disease in a non-Wuhan area of Hubei Province, China: a retrospective study.

BACKGROUND: Since December 2019, the 2019 coronavirus disease (COVID-19) has expanded to cause a worldwide outbreak that more than 600,000 people infected and tens of thousands died. To date, the clinical characteristics of COVID-19 patients in the non-Wuhan areas of Hubei Province in China have not been described. METHODS: We retrospectively analyzed the clinical characteristics and treatment progress of 91 patients diagnosed with COVID-19 in Jingzhou Central Hospital. RESULTS: Of the 91 patients diagnosed with COVID-19, 30 cases (33.0%) were severe and two patients (2.2%) died. The severe disease group tended to be older (50.5 vs. 42.0 years; p = 0.049) and have more chronic disease (40% vs. 14.8%; p = 0.009) relative to mild disease group. Only 73.6% of the patients were quantitative polymerase chain reaction (qPCR)-positive on their first tests, while typical chest computed tomography images were obtained for each patient. The most common complaints were cough (n = 75; 82.4%), fever (n = 59; 64.8%), fatigue (n = 35; 38.5%), and diarrhea (n = 14; 15.4%). Non-respiratory injury was identified by elevated levels of aspartate aminotransferase (n = 18; 19.8%), creatinine (n = 5; 5.5%), and creatine kinase (n = 14; 15.4%) in laboratory tests. Twenty-eight cases (30.8%) suffered non-respiratory injury, including 50% of the critically ill patients and 21.3% of the mild patients. CONCLUSIONS: Overall, the mortality rate of patients in Jingzhou was lower than that of Wuhan. Importantly, we found liver, kidney, digestive tract, and heart injuries in COVID-19 cases besides respiratory problems. Combining chest computed tomography images with the qPCR analysis of throat swab samples can improve the accuracy of COVID-19 diagnosis.

Neutrophils injure gallbladder interstitial Cajal-like cells in a guinea pig model of acute cholecystitis.

Acute cholecystitis is a common disease with gallbladder dysmotility. Disease pathogenesis involves immune cell infiltration as well as changes in gallbladder interstitial Cajal-like cells (ICLCs). However, it remains unclear if or how the immune cells affect ICLC morphology, density, distribution, and function in gallbladder tissue during acute cholecystitis. In this study, we explored the acute cholecystitis-related alterations in gallbladder ICLCs in a guinea pig model, focusing on the effects of neighboring neutrophils. Adult guinea pigs were randomly divided into four groups (control, 24 hr common bile duct ligation [CBDL], 48-hr CBDL, and antipolymorphonuclear neutrophil [PMN] treated) and analyzed using methylene blue staining and immunofluorescence. Gallbladder contractility was also monitored. To culture gallbladder ICLCs, collagenase digestion was performed on tissue from 10- to 15-day-old guinea pigs. Neutrophils isolated from the peripheral blood of experimental animals 48-hr postsurgery were also cocultured with the gallbladder ICLCs. Intracellular calcium was detected with Fluo-4 AM dye. Our results showed that gallbladder ICLC density significantly declined during acute cholecystitis and was accompanied by shortening of the cellular processes and damage to their network-like structure. However, pretreatment with anti-PMN partially prevented these changes. Gallbladder contraction was also significantly decreased during acute cholecystitis, and this appeared to be mediated by the neutrophils. Moreover, ICLCs cocultured with neutrophils also had shortened and reduced processes and impaired network-like structure formation. Intracellular calcium transient was less sensitive to contraction agonists and inhibitors when cocultured with neutrophils. Taken together, neutrophils greatly affect gallbladder ICLCs and dysmotility during acute cholecystitis.

Acute Cholecystitis Reduces Interstitial Cells of Cajal in Porcine Gallbladder Through Decreased mRNA Synthesis.

BACKGROUND/AIMS: Acute cholecystitis is a common gastrointestinal disorder, often characterized by acute cholecystitis with gallbladder motility disorder. Interstitial cells of Cajal (ICCs) are the pacemaker cells of gut motility in the gastrointestinal tract. Disruption of ICC function is related to motility disorders. The aim of this study was to explore the cellular and molecular mechanisms of ICCs in acute cholecystitis and after the resolution of acute inflammation. MATERIALS AND METHODS: Fifty adult guinea pigs were randomly divided into five groups: a sham-administered group (control group); two groups that were intraperitoneally administered an anti-polyclonal neutrophil (PMN) antibody 24 h before common bile duct ligation (CBDL); and two groups of guinea pigs that were subjected to CBDL without receiving the PMN antibody. Guinea pigs that underwent CBDL were held for 24 h or 48 h after surgery before being subjected to laparotomy and cholecystectomy. Immunohistochemistry, TUNEL assays, western blotting, and real-time PCR were performed to determine ICC morphology and density, to detect ICC apoptosis, and to examine stem cell factor (SCF) and c-kit protein expression and SCF and c-kit mRNA levels, respectively. RESULTS: Both hematoxylin-eosin staining and histological inflammation scores in the PMN groups were lower than those in the control groups (P < 0.01). No differences were observed in ICC morphology between groups. During acute cholecystitis, ICCs numbers were reduced. Conversely, the density of ICCs increased after inflammation was relieved (P < 0.01). In addition, SCF and c-kit protein and mRNA expression levels decreased during acute cholecystitis (P < 0.05) and increased after inflammation was relieved (P < 0.05). Furthermore, ICC apoptosis increased during acute cholecystitis and decreased after resolution of acute cholecystitis (P < 0.01). CONCLUSIONS: In acute cholecystitis, ICC injury may be related to gallbladder motility disorder.

Colonic Hypermotility in a Rat Model of Irritable Bowel Syndrome Is Associated with Upregulation of TMEM16A in Myenteric Plexus.

BACKGROUND: Irritable bowel syndrome (IBS) is a common disease with intestinal dysmotility, whose mechanism remains elusive. TMEM16A is a calcium-activated chloride channel (CaCC) involved in intestinal slow-wave generation. AIMS: To investigate whether TMEM16A is involved in colonic dysmotility in IBS. METHODS: A rat model of IBS was established by chronic water avoidance stress (WAS). Colonic pathological alterations were evaluated histologically, and intestinal motility was assessed by intestinal transit time (ITT) and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response (VMR) to colorectal distension (CRD). TMEM16A expression was evaluated by RT-PCR, Western blot, and immunofluorescence. Colonic muscle strip contractility was measured by isometric transducers, and the effect of niflumic acid (NFA), a CaCC antagonist, on colonic motility was examined. RESULTS: After 10 days of WAS exposure, ITT was decreased and FWC was elevated. Furthermore, VMR magnitude of WAS rats in response to CRD was significantly enhanced. Protein and mRNA levels of TMEM16A in colon were considerably increased after WAS. The percentage of TMEM16A-positive neurons in myenteric plexus (MP) of WAS rats was significantly higher than controls. Pharmacological blockade of TMEM16A activity by NFA considerably enhanced ITT, with concentration-dependent declines in FWC and VMR magnitude in NFA-treated rats. Further, spontaneous contraction of colonic strips of NFA-treated rats was significantly ameliorated in a concentration-dependent manner in vitro. CONCLUSIONS: Upregulation of TMEM16A in MP neurons may play an important role in chronic stress-induced colonic hypermotility, making CaCC-blocking drugs a putatively effective treatment method for colonic hypermotility in IBS.

Effect of Neutrophils on Gallbladder Interstitial Cajal-Like Cells in Guinea Pig Model of Acute Cholecystitis.

BACKGROUND: Acute cholecystitis is a common condition in gallbladder motility disorder. Interstitial Cajal-like cells (ICLCs) in the gallbladder are known as one of the players in the complex motility mechanisms affecting gallbladder motility. AIM: This study explored morphological symptoms and molecular mechanisms underlying gallbladder ICLC changes induced by acute cholecystitis. MATERIALS AND METHODS: Fifteen adult guinea pigs were randomly divided into 3 groups: sham-operated group (healthy controls) and 2 experimental groups wherein these guinea pigs were subjected to common bile duct ligation to induce acute cholecystitis. Neutrophils were isolated from the peripheral blood of sham-operated animals and from the experimental animals at 24 and 48 h after surgery, and co-cultured with gallbladder ICLCs. The morphology of gallbladder ICLCs was examined by laser confocal immunofluorescence microscopy, TUNEL assay was used to detect apoptosis, and western blot and real-time PCR were performed to detect stem cell factor (SCF) and c-kit protein and mRNA expression, respectively. RESULTS: No morphological differences in the gallbladder ICLCs were observed between single-culture and co-culture with healthy control neutrophil groups. However, the ICLCs in all co-culture groups with acute inflammation were impaired. In the co-culture groups, the rate of ICLC apoptosis was significantly higher than that in the single-culture group. SCF and c-kit protein and mRNA expression levels decreased in all co-culture groups as well. CONCLUSION: We demonstrated that the neutrophils are involved in gallbladder ICLC injury in acute cholecystitis cases and associated with gallbladder motility disorder.

The Role of Interstitial Cells of Cajal in Acute Cholecystitis in Guinea Pig Gallbladder.

BACKGROUND/AIMS: Acute cholecystitis is common in gallbladder motility disorder. Interstitial cells of Cajal (ICCs) in the gallbladder are involved in the regulation of gallbladder motility. The aim of this study was to explore the change of gallbladder ICCs in acute cholecystitis. METHODS: Thirty adult guinea pigs were randomly divided into 3 groups: a sham-operated group (healthy controls) and 2 study groups. The animals in the study group were subjected to bile duct ligation and then to laparotomy and cholecystectomy at 24 and 48 hours after surgery. Immunohistochemistry, immunohistofluorescence, and laser confocal microscopy were performed to observe the shape, size, morphology, and density of gallbladder ICCs. Western blot and real-time PCR were performed to detect stem cell factor and c-kit protein and mRNA expression, respectively. RESULTS: There were no differences in the shape, size, and morphology of the gallbladder ICCs in the control and the two acute cholecystitis groups. Density of gallbladder ICCs, SCF level, and c-kit protein and mRNA expression all decreased in the acute cholecystitis groups. Further, SCF level and c-kit protein and mRNA expression decreased with progress of acute cholecystitis (all P < 0.05). CONCLUSION: Acute cholecystitis can decrease ICCs through repression of SCF and c-kit expression and that ICCs loss play a role in acute cholecystitis.

Fusobacterium nucleatum promotes esophageal squamous cell carcinoma progression and chemoresistance by enhancing the secretion of chemotherapy-induced senescence-associated secretory phenotype via activation of DNA damage response pathway.

Senescence frequently occurs in cancer cells in response to chemotherapy (called therapy-induced senescence). Senescent cells can exert paracrine effects through the senescence-associated secretory phenotype (SASP) promoting cancer recurrence and chemoresistance. The altered gut microbiota has been closely associated with cancer progression through the direct interaction with cancer cells. However, little is known about the relationship between the gut microbiota and therapy-induced senescent cells. This study aimed to explore the impact of the gut microbiota on therapy-induced senescent cells and the SASP. We found that esophageal squamous cell carcinoma (ESCC) cells were induced into senescence following platinum-based chemotherapy, accompanied by the secretion of a robust SASP. Furthermore, senescent ESCC cells exerted a tumor-promoting effect through the SASP both in vitro and in vivo. Through 16S rRNA gene sequencing and fluorescence in situ hybridization, we identified that Fusobacterium nucleatum (F. nucleatum) was abundant in human ESCC cancerous tissues and correlated with poor prognosis in ESCC patients. Notably, F. nucleatum further promoted the secretion of the SASP by senescent ESCC cells. Compared with the conditioned medium from senescent ESCC cells, the conditioned medium from F. nucleatum-treated senescent ESCC cells accelerated tumor growth in xenograft models, enhanced migration and invasion abilities, and potentiated chemoresistance both in vitro and in vivo. Mechanistically, F. nucleatum invaded and survived in senescent ESCC cells and induced an increase in DNA damage to further activate the DNA damage response pathway, thus enhancing the SASP. Altogether, these findings reveal for the first time that F. nucleatum promotes the secretion of chemotherapy-induced SASP to drive ESCC progression and chemoresistance, which supports F. nucleatum as a potential target for ESCC therapy.

Senescent cells in cancer therapy: why and how to remove them.

Cellular senescence is a stress response that imposes a growth arrest on cancer and nonmalignant cells during cancer therapy. By secreting a plethora of proinflammatory factors collectively termed the senescence-associated secretory phenotype (SASP), therapy-induced senescent cells can promote tumorigenesis. Moreover, the SASP from senescent cells is also able to drive therapy resistance and mediate many adverse effects of cancer therapy. Because senescent cell production often occurs during cancer therapy, it is important to carefully consider these potential detrimental effects. Senotherapy, which refers to selective removal of senescent cells, has been proposed as a promising adjuvant approach to eliminate the adverse effects of senescent cells. Thus, in this review we summarize in detail the mechanisms by which senescent cells contribute to tumorigenesis and therapeutic resistance. Also, we thoroughly discuss the potential strategies regarding how to effectively circumvent the undesirable effects of therapy-induced senescent cells.

[Significance of caveolin-1 protein detected by quantum dots technique in human lung cancer invasiveness and metastasis.].

OBJECTIVE: Fluorescent semiconductor nanocrystals [also known as quantum dots (QDs)] are nanometer-sized light-emitting particles and are emerging as a new class of fluorescent probes for cancer detection, due to their unique optical and electronic properties. The aim of this study was to investigate the expression of caveolin-1 (Cav-1), extracellular matrix metalloproteinase inducer (CD(147)/EMMPRIN), matrix metalloproteinase-2 (MMP-2) proteins in the human lung cancer tissue microarray (TMA) by QDs immunofluorescence histochemistry (QDs-IHC) and therefore to evaluate the relationship between Cav-1 protein and lung cancer invasiveness and metastasis. METHODS: QDs-IHC combined with TMA were used to detect the expression of Cav-1, CD(147) and MMP-2 proteins in 70 cases of human lung cancers and 5 cases of noncancerous lung tissues. RESULTS: The average immunofluorescence intensity of Cav-1 protein in the lung cancer group was 55 +/- 23, significantly lower than that in the control group (80 +/- 4, t = 2.461, P = 0.016). The expression of Cav-1 was not associated with the age and the gender of the patients, nor with the histology type of lung cancer (P > 0.05). The average immunofluorescence intensity of Cav-1 protein was associated significantly with TNM staging (t = 2.466, P = 0.016) and lymph node metastasis (t = 2.972, P = 0.004). A negative correlation was observed between Cav-1 and CD(147) protein expression (r = -0.331, P = 0.005), but no correlation was observed between Cav-1 and MMP-2 protein expression (P = 0.193). CONCLUSIONS: QDs-IHC could accurately and quantitatively detect different protein location in lung cancer TMA. A close relationship was detected between Cav-1 protein and the development of lung cancer. High expression of Cav-1 may be involved in invasiveness and metastasis of lung cancer, possibly through the regulation of CD(147) rather than MMP-2 activition.

Emodin increases Ca(2+) influx through L-type Ca(2+) channel in guinea pig gallbladder smooth muscle.

Emodin is known to be used in the treatment of cholesterol stones and cholecystitis. This study sought to investigate the effects of emodin on the contraction of gallbladder smooth muscle (GBSM), intracellular Ca(2+) concentration and L-type calcium current in GBSM cells. Gallbladder muscle strips were obtained from adult guinea pigs and the resting tension was recorded. Gallbladder smooth muscle cells were isolated by enzymatic digestion. Cells were loaded with fluo-3/AM and [Ca(2+)](i) was determined by a laser confocal microscope. Calcium current was recorded by the whole-cell patch clamp method. Emodin increased the resting tension of GBSM strips in a dose-dependent manner. Emodin elevated [Ca(2+)](i) in GBSM cells, and this effect was attenuated by pretreatment with nifedipine. In addition, Emodin increased L-type calcium current at concentrations of 1 to 30 microM (at +10 mV, 10 microM, 45.1+/-5.2% compared to control, EC(50) =3.11 microM). In the presence of protein kinase C (PKC) inhibitor, Staurosporine, emodin did not significantly affect the calcium current. However, phorbol 12, 13-dibutyrate mimicked emodin in enhancement of the calcium current. These results suggest that emodin promotes gallbladder contraction by increasing Ca(2+) influx through L-type calcium channel via PKC pathway.

Control of gallbladder contractions by cholecystokinin through cholecystokinin-A receptors on gallbladder interstitial cells of Cajal.

AIM: To identify the cholecystokinin (CCK)-A receptors (CCK-AR) on the guniea pig gallbladder interstitial cells of cajal (ICC) and to study CCK-8 induced gallbladder muscle strip contractions through the CCK-AR. METHODS: The existence of CCK-AR was examined by immunohistofluorescence on sectioned tissue and cultured cells. In vitro contractile response of guinea pig gallbladder muscle strips and the strips with ICC removed were also studied with CCK-8 receptors added. RESULTS: In tissue sections, intensely CCKAR-immunoreactive interstitial cells were found mainly in the muscular layers. In cultured cell sections, distinctive double staining of C-kit and CCK-AR ICCs were found. When we removed the ICC of the gallbladder, CCK-8 induced muscle strip contraction dose response curve significantly shifted to the right. CONCLUSION: We proved that both the existence of CCK-AR on the guinea pig gallbladder ICC and CCK evoked contraction are mediated through direct action on CCK-AR on the gallbladder ICC.

Role of High-affinity Choline Transporter 1 in Colonic Hypermotility in a Rat Model of Irritable Bowel Syndrome.

BACKGROUND/AIMS: Irritable bowel syndrome (IBS) is a common disease characterized by intestinal dysmotility, the mechanism of which remains elusive. We aim to determine whether the high-affinity choline transporter 1 (CHT1), a determinant of cholinergic signaling capacity, modulates intestinal motility associated with stress-induced IBS. METHODS: A rat IBS model was established using chronic water avoidance stress (WAS). Colonic pathological alterations were evaluated histologically and intestinal motility was assessed by intestinal transit time and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response to colorectal distension. RT-PCR, western blotting, and immunostaining were performed to identify colonic CHT1 expression. Contractility of colonic muscle strips was measured using isometric transducers. enzyme-linked immunosorbent assay was used to measure acetylcholine (ACh). We examined the effects of MKC-231, a choline uptake enhancer, on colonic motility. RESULTS: After 10 days of WAS, intestinal transit time was decreased and fecal water content increased. Visceromotor response magnitude in WAS rats in response to colorectal distension was significantly enhanced. Protein and mRNA CHT1 levels in the colon were markedly elevated after WAS. The density of CHT1-positive intramuscular interstitial cells of Cajal and myenteric plexus neurons in WAS rats was higher than in controls. Ammonium pyrrolidine dithiocarbamate partly reversed CHT1 upregulation and alleviated colonic hypermotility in WAS rats. Pharmacological enhancement of CHT1 activity by MKC-231 enhanced colonic motility in control rats via upregulation of CHT1 and elevation of ACh production. CONCLUSION: Upregulation of CHT1 in intramuscular interstitial cells of Cajal and myenteric plexus neurons is implicated in chronic stress-induced colonic hypermotility by modulation of ACh synthesis via nuclear factor-kappa B signaling.

Upregulation of the high-affinity choline transporter in colon relieves stress-induced hyperalgesia.

BACKGROUND: Irritable bowel syndrome (IBS) is a common disease with hyperalgesia, the mechanisms of which remain elusive. The cholinergic system is known to be involved in pain inhibitory pathways in multiple diseases, and its involvement in IBS is unknown. OBJECTIVE: We aimed to determine whether high-affinity choline transporter CHT1, a major determinant of the cholinergic signaling capacity, is involved in regulating intestinal sensations associated with stress-induced visceral pain. MATERIALS AND METHODS: An IBS rat model was established by chronic water avoidance stress (WAS). Colonic pathologic alterations were detected by H&E staining. Visceral sensations were determined by scoring the abdominal withdrawal reflex (AWR) and visceromotor response (VMR) magnitude of the electromyogram in response to colorectal distension (CRD). Abdominal mechanical hyperalgesia was assessed by counting the number of withdrawal events evoked by applying von Frey filaments. Real-time PCR, Western blot, and immunostaining were performed to identify CHT1 expression in the colon. Acetylcholine (ACh) secretion was determined by ELISA. Effects of MKC-231, a choline uptake enhancer, on visceral pain were examined. RESULTS: After 10 days of WAS exposure, AWR score and VMR magnitude in response to CRD were significantly enhanced and the number of withdrawal events was elevated. Protein and mRNA levels of CHT1 were considerably increased in the colon after WAS. CHT1 upregulation in the WAS-exposed group was largely abolished by ammonium pyrrolidinedithiocarbamate. The density of CHT1-positive intramuscular cells and enteric neurons in the myenteric plexus was enhanced in WAS-exposed rats. Pharmacologic enhancement of CHT1 activity by MKC-231 gavage could relieve the visceral pain of WAS rats by upregulating CHT1 protein expression and enhancing ACh production. CONCLUSION: CHT1 may exert an antinociceptive effect in stress-induced visceral pain by modulating ACh synthesis through nuclear factor kappa B signaling. MKC-231 could be used as a potential drug to treat disorders with hyperalgesia.

Distribution changes of interstitial cells of Cajal during cholesterol gallstone formation in guinea pigs fed a high cholesterol diet.

BACKGROUND: Cholesterol gallstone is commonly observed in patients with gallbladder disorders. Interstitial cells of Cajal (ICCs) in the gallbladder are important for regulating gallbladder motility and have a close relationship with cholelithiasis. AIM: The aim of this study was to explore changes in the distribution of gallbladder ICCs during cholesterol gallstone formation. MATERIALS AND METHODS: Thirty guinea pigs were randomly divided into three groups: the control group and study groups. Animals in study groups were fed on high cholesterol diet for 4 weeks or 8 weeks. Animals in the control groups were fed on a standard diet for 8 weeks. Immunohistochemistry was performed to observe the shape, size, morphology, and numbers of ICCs from the neck of the gallbladder to the fundus of the gallbladder, and terminal deoxynucleotidyl transferase dUTP nick-end labeling was performed to detect apoptosis in ICCs from the upper part of the gallbladder to the lower part of the gallbladder. RESULTS: There were no differences in the shape, size, and morphology of the gallbladder ICCs in all groups. Cholesterol gallstones formed in guinea pigs fed on high cholesterol diet. The numbers of gallbladder ICCs were significantly decreased from the neck of the gallbladder to the fundus of the gallbladder, and gallbladder ICC apoptosis was significantly increased from the upper part of the gallbladder to the lower part of the gallbladder in both guinea pigs fed on high cholesterol diet (all P<0.05). CONCLUSION: Cholesterol gallstone formation reduced the density of gallbladder ICCs and increased the frequency of apoptotic gallbladder ICCs from the neck of the gallbladder to the fundus of the gallbladder, and these alterations may affect gallbladder ICC function.

Expression of NF-kappaB and human telomerase reverse transcriptase in gastric cancer and precancerous lesions.

AIM: To investigate the expression of NF-kappaBp65 protein and human telomerase reverse transcriptase (hTERT) and their correlation in gastric cancer and precancerous lesions. METHODS: Forty-one patients with primary gastric cancer, 15 with dysplasia, 23 intestinal metaplasia and 10 with normal gastric mucosa were included in this study. Expression of NF-kappaBp65 protein, hTERT mRNA and protein were determined by immunohistochemistry and in situ hybridization. RESULTS: The rate of p65 expression in normal gastric mucosa, intestinal metaplasia, dysplasia and carcinoma was 0%, 34.78%, 53.33% and 60.98%, respectively, while the rate of hTERT mRNA expression was 10.00%, 39.13%, 66.67% and 85.37% and the rate of hTERT protein expression was 0%, 30.43%, 60.00% and 78.05%, respectively. All the three parameters were significantly increased in dysplasia and carcinoma compared to normal mucosa, while the expression levels were also significantly higher in carcinoma than in intestinal metaplasia (P<0.05). In gastric cancer tissues, nuclear staining rates of p65 and hTERT protein were both significantly associated with the degree of differentiation, lymph node metastasis, clinical stage and invasion depth (P<0.05). However, hTERT mRNA expression was only significantly associated with clinical stage. There was a positive correlation between p65 and hTERT mRNA (rs=0.661-0.752, P<0.01), and between hTERT protein and hTERT mRNA (rs=0.609-0.750, P<0.01). CONCLUSION: NF-kappaBp65 and hTERT expressions are upregulated at the early stage of gastric carcinogenesis. NF-kappaB activation may contribute to hTERT expression and thereby enhance telomerase activity, which represents an important step in carcinogenesis progress.

Preventive effect of hydrotalcite on gastric mucosal injury in rats induced by taurocholate.

AIM: To study the preventive effect of hydrotalcite on gastric mucosal injury in rat induced by taurocholate, and to investigate the relationship between the protective mechanism of hydrotalcite and the expression of trefoil factor family 2 (TFF2) mRNA and c-fos protein. METHODS: Forty five male Wistar rats were randomly divided into hydrotalcite group, ranitidine group and control group. Gastric mucosal injury was induced by introgastric acidified taurocholate. OD value of TFF2 mRNA expression in gastric mucous cells was determined by hybridization and computer image analysis system. OD value of c-fos protein expression in gastric mucous cells was measured by immunohistochemistry and computer image analysis system. RESULTS: The gross mucosal injury index in hydrotalcite group was significantly lower than that in ranitidine group and control group (8.60+/-2.20 vs 16.32+/-4.27, 29.53+/-5.39; P<0.05, P<0.01). The expression level of TFF2 mRNA in hydrotalcite group was markedly higher than that in ranitidine group and control group (0.56+/-0.09 vs 0.30+/-0.05, 0.28+/-0.03, P<0.05). The OD value of c-fos protein in hydrotalcite group was higher than that in ranitidine group and control group (0.52+/-0.07 vs 0.31+/-0.04, 0.32+/-0.05, P<0.05). CONCLUSION: Hydrotalcite can protect gastric mucosal injury in rats induced by taurocholate, which may be related to the increased expression of TFF2 and c-fos protein.

Detection of type IV collagenase activity in malignant ascites.

AIM: Type IV collagenase participates in invasion and metastasis of cancer cells. Malignant ascites is a manifestation of advanced malignant disease that is associated with invasion and metastasis of the peritoneal cavity. Thus, it is reasonable to hypothesize that type IV collagenase is linked to malignant ascites. The purpose of our study was to detect type IV collagenase activity in malignant ascites so as to provide the scientific basis for clinic diagnosis and treatment of malignant ascites. METHODS: Cirrhotic ascites (n=36), tuberculous ascites (n=8) and malignant ascites (n=23) from patients with gastric cancer (n=6), colon cancer (n=5), ovarian cancer (n=8) and other cancers (n=4), including 2 hepatocellular cancers, 1 pancreatic cancer, 1 primary peritoneal carcinoma were collected by paracentesis. The ascites were made cell-free by centrifugation and stored frozen at -70 degrees C before determination. Type IV collagenase activity was determined by gelatin zymography. RESULTS: The activity of matrix metalloproteinases-2 and -9 could not be detected in ascites of hepatic cirrhosis and tuberculous peritonitis but could be detected in 20 and 18 out of 23 malignant ascites respectively. The positive rate of type IV collagenase (MMP-2, 87.0% and MMP-9, 78.3%) was higher than that by routine ascites tests (P<0.01) in malignant ascites. Furthermore, the activity of MMP-2 was higher than that of MMP-9 (P=0.022<0.05). CONCLUSION: Type IV collagenase is positive in malignant ascites. Detection of type IV collagenase activity is useful in qualitative diagnosis of ascites. Type IV collagenase may play an important role in malignant ascites formation.

Role of VEGF and CD44v6 in differentiating benign from malignant ascites.

AIM: To detect the vascular endothelial growth factor (VEGF) and soluble splice variant 6 of CD44 (sCD44v6) levels in ascites and to explore their role in differentiating benign from malignant ascites. METHODS: Cirrhotic ascites (n=36), tuberculosis ascites (n=8) and malignant ascites (n=23) were collected and studied. Concentrations of soluble VEGF and sCD44v6 in various kinds of ascites (n=67) were measured using a sandwich enzyme-linked immunoadsorbent assay. RESULTS: VEGF and sCD44v6 levels in malignant ascites were 640.74+/-264.81 pg/ml and 89.22+/-38.20 ng/ml, respectively, both of which were significantly higher than those in cirrhotic ascites and tuberculous ascites (q=18.98, 11.89 and q=8.92, 5.09; P<0.01). However, the levels of VEGF and sCD44v6 in cirrhotic and tuberculous ascites had no significant difference (q=0.48, 0.75; P>0.05). Furthermore, VEGF levels in malignant ascites in patients with ovarian cancer were higher than those with gastric and colon cancer (q=5.03, 6.79; P<0.01, respectively). But differences of VEGF levels between gastric and colon cancer were not significant (q=1.90, P>0.05). Whereas, sCD44v6 levels in malignant ascites from patients with ovarian, gastric and colon cancer had no significant difference (q=0.06, 0.91, 0.35; P>0.05, respectively). In comparison with cirrhotic and tuberculous ascites, when the upper limit of its VEGF mean levels 119.44 pg/ml (70.90+/-48.54) and sCD44v6 mean levels 63.59 ng/ml (44.42+/-19.17) was taken as the minimum cutoff limit, the sensitivity and specificity of VEGF and sCD44v6 of this assay to the diagnosis of malignant ascites were 91.3%, 90.9% and 73.9%, 88.7% respectively. CONCLUSION: Elevated levels of VEGF and sCD44v6 may be useful in differential diagnosis of benign and malignant ascites.