Hydrogen-rich water protects against acetaminopheninduced hepatotoxicity in mice

AIM: To investigate the hepatoprotective effects and mechanisms of hydrogen-rich water(HRW) in acetaminophen(APAP)-induced liver injury in mice.METHODS: Male mice were randomly divided into the following four groups: normal saline(NS) control group, mice received equivalent volumes of NS intraperitoneally(ip); HRW control group, mice were given HRW(same volume as the NS group); APAP + NS group, mice received NS ip for 3 d(5 mL /kg body weight, twice a day at 8 am and 5 pm) after APAP injection; APAP + HRW group, mice received HRW for 3 d(same as NS treatment) after APAP challenge.In the first experiment, mice were injected ip with a lethal dose of 750 mg/kg APAP to determine the 5-d survival rates.In the second experiment, mice were injected ip with a sub-lethal dose of 500 mg/kg.Blood and liver samples were collected at 24, 48, and 72 h after APAP injection to determine the degree of liver injury.RESULTS :Treatment with HRW resulted ina significant increase in the 5-d survival rate compared with the APAP + NS treatment group(60% vs 26.67%, P 0.05).HRW could significantly decrease the serum alanine aminotransferase level(24 h: 4442 ± 714.3 U/L vs 6909 ± 304.8 U/L, P 0.01; 48 h: 3782 ± 557.5 U/L vs 5111 ± 404 U/L, P 0.01; and3255 ± 337.4 U/L vs 3814 ± 250.2 U/L, P 0.05, respectively) and aspartate aminotransferase level(24 h: 4683 ± 443.4 U/L vs 5307 ± 408.4 U/L, P 0.05; 48 h: 3392 ± 377.6 U/L vs 4458 ± 423.6 U/L, P 0.01; and 3354 ± 399.4 U/L vs 3778 ± 358 U/L, respectively) compared with the APAP treatment group.The alkaline phosphatase, total bilirubin and lactate dehydrogenase levels had the same result.Seventy-two hours after APAP administration, liver samples were collected for pathological examination and serum was collected to detect the cytokine levels.The liver index(5.16% ± 0.26% vs 5.88% ± 0.073%, P 0.05) and percentage of liver necrosis area(27.73% ± 0.58% vs 36.87% ± 0.49%, P 0.01) were significantly lower in the HRW-treated animals.The malonyldialdehyde(MDA) contents were significantly reduced in the HRW pretreatment group, but they were increased in the APAP-treated group(10.44 ± 1.339 nmol/mg protein vs 16.70 ± 1.646 nmol/mg protein, P 0.05).A decrease in superoxide dismutase(SOD) activity in the APAP treatment group and an increase of SOD in the HRW treatment group were also detected(9.74 ± 0.46 U/mg protein vs 12.1 ± 0.67 U/mg protein, P 0.05).Furthermore, HRW could significantly increase the glutathione(GSH) contents(878.7 ± 76.73 mg/g protein vs 499.2 ± 48.87 mg/g protein) compared with the APAP treatment group.Meanwhile, HRW could reduce the inflammation level(serum TNF-α: 399.3 ± 45.50 pg/L vs 542.8 ± 22.38 pg/L, P 0.05; and serum IL-6: 1056 ± 77.01 pg/L vs 1565 ± 42.11 pg/L, P 0.01, respectively).In addition, HRW could inhibit 4-HNE, nitrotyrosine formation, JNK phosphorylation, connexin 32 and cytochrome P4502 E expression.Simultaneously, HRW could facilitate hepatocyte mitosis to promote liver regeneration.CONCLUSION: HRW has significant therapeutic potential in APAP-induced hepatotoxicity by inhibiting oxidative stress and inflammation and promoting liver regeneration.     

Xiaotan Tongfu granules contribute to the prevention of stress ulcers

AIM:To investigate the efficacy and potential mechanism of Xiaotan Tongfu granules(XTTF)in stress ulcers.METHODS:One hundred sixty rats were randomly divided into 4 groups(n=10)as follows:the model group(MP group),the control group(CP group),the ranitidine group(RP group)and the XTTF granule group(XP group).Rats in the MP group received no drugs,rats in the CP group received 0.2 mL of a 0.9%sodium chloride solution via oral gavage,and rats in the RP and XP groups received the same volume of ranitidine(50 mg/kg)or XTTF granule(4.9 g/kg).The cold-restraint stress model was applied to induce stress ulcers after 7 consecutive days of drug administration.Afterwards,rats were sacrificed at 0,3,6 and24 h.Gastric pH was measured by a precise pH meter;gastric emptying rate(GER)was measured by using a methylcellulose test meal;myeloperoxidase activity(MPO),macrophage migration inhibitory factor(MIF),proliferating cell nuclear antigen(PCNA),and heat shock protein 70(HSP70)were measured by immunohistochemical staining;and mucosal cell apoptosis was measured by transferase dUTP nick end labeling.RESULTS:In the cold-restraint stress model,the development of stress ulcers peaked at 3 h and basically regressed after 24 h.Gastric lesions were significantly different in the RP and XP groups at each time point.Interestingly,although this index was much lower in the RP group than in the XP group immediately following stress induction(7.00±1.10 vs 10.00±1.79,P<0.05.Concerning gastric pH,between the RP and XP groups,we detected a statistically significant difference immediately after stress induction(0 h:4.56±0.47 vs 3.34±0.28,P<0.05)but not at any of the subsequent time points.For GER,compared to the RP group,GER was remarkably elevated in the XP group because a statistically significant difference was detected(3 h:46.84±2.70 vs 61.16±5.12,P<0.05;6 h:60.96±6.71 vs 73.41±6.16,P<0.05;24 h:77.47±3.17 vs 91.31±4.34,P<0.05).With respect to MPO and MIF,comparisons between the RP and XP groups revealed statisticall     

Roles of BN52021 in platelet-activating factor pathway in inflammatory MS1 cells

AIM: To determine the effects of BN52021 on platelet-activating factor receptor (PAFR) signaling molecules under lipopolysaccharide (LPS)-induced inflammatory conditions in MS1 cells. METHODS: MS1 cells (a mouse pancreatic islet endothelial cell line) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine and 100 μg/mL penicillin/streptomycin in 5% CO 2 at 37 ℃. After growth to confluency in media, the cells were processed for subsequent studies. The MS1 cells received 0, 0.1, 1 and 10 μg/mL LPS in this experiment. The viability/prolifera-tion of the cells induced by LPS was observed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Apoptosis and necrosis of the cells under the inflammatory condition described previously were observed using Hoechst 33342-propidium iodide staining. Adenylate cyclase (AC), phospholipase A 2 (PLA 2 ), phospholipase Cβ (PLCβ), protein tyrosine kinase (PTK), G protein-coupled receptor kinases (GRK) and p38-mitogen-activated protein kinase (p38 MAPK) mRNA in the PAFR signaling pathway were measured by real-time polymerase chain reaction. The protein expression level of phosphorylated AC (p-AC), phosphorylated PLA 2 (p-PLA 2 ), phosphorylated PTK (p-PTK), phosphorylated p38 MAPK (p-p38 MAPK), PLCβ and GRK was measured using Western blotting analysis. RESULTS: The activity of MS1 cells incubated with dif- ferent concentrations of LPS for 6 h decreased significantly in the 1 μg/mL LPS group (0.49 ± 0.10 vs 0.67 ± 0.13, P < 0.05) and 10 μg/mL LPS group (0.44 ± 0.10 vs 0.67 ± 0.13, P < 0.001), but not in 0.1 μg/mL group. When the incubation time was extended to 12 h (0.33 ± 0.05, 0.32 ± 0.03 and 0.25 ± 0.03 vs 0.69 ± 0.01) and 24 h (0.31 ± 0.01, 0.29 ± 0.03 and 0.25 ± 0.01 vs 0.63 ± 0.01), MS1 cell activity decreased in all LPS concentration groups compared with the blank control (P < 0.001). BN52021 significantly improved the cell activity when its concentration     

Intestinal dendritic cells change in number in fulminant hepatic failure

AIM:To investigate the change in intestinal dendritic cell(DC)number in fulminant hepatic failure(FHF).METHODS:An animal model of FHF was created.Intestinal CD11b/c was detected by immunohistochemistry and Western blot.Quantitative real-time polymerase chain reaction(PCR)was used to detect intestinal integrin-αm RNA expression.Intestinal CD83,CD86,CD74,CD3 and AKT were detected by immunohistochemistry,Western blot and PCR.Phosphorylated-AKT(p-AKT)was detected by immunohistochemistry and Western blot.RESULTS:In the FHF group[D-galactosamine(D-Galn)+lipopolysaccharide(LPS)group],the mice began to die after 6 h;conversely,in the D-Galn and LPS groups,the activity of mice was poor,but there were no deaths.Immunohistochemistry results showed that in FHF,the expression of CD11b/c(7988400±385941vs 1102400±132273,P0.05),CD83(13875000±467493 vs 9257600±400364,P0.05),CD86(7988400±385941 vs 1102400±13227,P0.05)and CD74(11056000±431427 vs 4633400±267903,P0.05)was significantly increased compared with the normal saline(NS)group.Compared with the NS group,the protein expression of CD11b/c(5.4817±0.77 vs 1.4073±0.37,P0.05)and CD86(4.2673±0.69 vs 1.1379±0.42,P0.05)was significantly increased.Itg-α(1.1224±0.3 vs 0.4907±0.19,P0.05),CD83(3.6986±0.40 vs 1.0762±0.22,P0.05)and CD86(1.5801±0.32 vs 0.8846±0.10,P0.05)m RNA expression was increased significantly in the FHF group.At the protein level,expression of CD74in the FHF group(2.3513±0.52)was significantly increased compared with the NS group(1.1298±0.33),whereas in the LPS group(2.3891±0.47),the level of CD74 was the highest(P0.05).At the gene level,the relative expression of CD74 m RNA in the FHF group(1.5383±0.26)was also significantly increased in comparison to the NS group(0.7648±0.22;P0.05).CD3 expression was the highest in the FHF group(P0.05).In the FHF,LPS and D-Galn groups,the expression of AKT at the protein and m RNA levels was elevated compared with the NS group,but there wasno statistical significance(P0.05).The p-AKT protein expression in the FHF(1.54±0.06),LPS(1.56±0.05)and D-Galn(1.29±0.03)groups was higher than that in the NS group(1.07±0.03)(P0.05).CONCLUSION:In FHF,a large number of DCs mature,express CD86,and activate MHC classⅡmolecular pathways to induce a T cell response,and the AKT pathway is activated.     

Multispecies probiotic protects gut barrier function in experimental models

AIM:To investigate the effect of the probiotic combination Lactibiane Tolerance?(LT)on epithelial barrier function in vitro and in vivo.METHODS:The effect of the multispecies probiotic LT was assessed on several models of epithelial barrier function both in vitro(in basal and inflammatory conditions)and in vivo[visceral hypersensitivity induced by chronic stress or by colonic perfusion of a fecal supernatant(FSN)from patients with irritable bowel syndrome(IBS)].In vitro,we measured the permeability of confluent T84 cell monolayers incubated with or without LT by evaluating the paracellular flux of macromolecules,in basal conditions and after stimulation with lipopolysaccharide(LPS)or with conditioned medium of colonic biopsies from IBS patients(IBS-CM).In vivo,male C57/Bl6 mice received orally NaCl or LT for 15 d and were submitted to water avoidance stress(WAS)before evaluating visceral sensitivity by measuring the myoelectrical activity of the abdominal muscle and the paracellular permeability with 51Cr-EDTA.Permeability and sensitivity were also measured after colonic instillation of FSN.Tight-junctions were assessed by immunoblotting and TLR-4 expression was evaluated by immunohistochemistry RESULTS:Incubation of T84 cell monolayers with LT in basal conditions had no significant effect on permeability(P>0.05 vs culture medium).By contrast,addition of LT bacterial bodies(LT)completely prevented the LPS-induced increase in paracellular permeability(P<0.01 vs LPS 10 ng/mL(LPS 10);P<0.01 vs LPS 100ng/mL(LPS 100),P>0.05 vs culture medium).The effect was dose dependent as addition of 109 LT bacterial bodies induced a stronger decrease in absorbance than 106 LT(109 LT+LPS 10:-20.1%±13.4,P<0.01vs LPS 10;106 LT+LPS 10:-11.6%±6.2,P<0.01 vs LPS 10;109 LT+LPS 100:-14.4%±5.5,P<0.01 vs LPS 100;106 LT+LPS 100:-11.6%±7.3,P<0.05 vs LPS 100).Moreover,the increase in paracellular permeability induced by culturing T84 cells with conditioned medium of colonic biopsies from IBS patients(IBS-CM)was completely inhibited in the presence of 109 LT(P<0.01 vs IBS-CM).LT also significantly prevented the epithelial disruption induced by intracolonic infusion of fecal supernatant from IBS patients(P<0.01 vs IBS FSN)or water avoidance stress P<0.01 vs WAS)in C57/Bl6 mice and increased the expression of occludin in vitro and in vivo,as assessed by immnunoblotting.The WAS-induced effect on visceral sensitivity was prevented by LT treatment since values obtained for all steps of colorectal distension were significantly(P<0.01)different from the WAS group.Finally,LT downregulated the response mediated through TLR-4 in vitro(decrease in tumor necrosis factorαsecretion in response to LPS:-65.8%for 109 LT and-52.5%for 106LT,P<0.01 vs LPS)and in vivo(inhibition of WAS induced an increase in TLR-4 expression in the LT treated mice colon,P<0.01 vs WAS).CONCLUSION:The probiotic LT mix prevented the disruption to the epithelial barrier induced by LPS,stress or colonic soluble factors from IBS patients and prevented visceral hypersensitivity.     

Effectiveness and safety of continuous wound infiltration for postoperative pain management after open gastrectomy

AIM: To prospectively evaluate the effectiveness and safety of continuous wound infiltration(CWI) for pain management after open gastrectomy. METHODS: Seventy-five adult patients with American Society of Anesthesiologists(ASA) Physical Status Classification System(ASA) grade 1-3 undergoing open gastrectomy were randomized to three groups. Group 1 patients received CWI with 0.3% ropivacaine(group CWI). Group 2 patients received 0.5 mg/m L morphine intravenously by a patient-controlled analgesia pump(PCIA)(group PCIA). Group 3 patients received epidural analgesia(EA) with 0.12% ropivacaine and 20 μg/m L morphine with an infusion at 6-8 m L/h for 48 h(group EA). A standard general anesthetic technique was used for all three groups. Rescue analgesia(2 mg bolus of morphine, intravenous) was given when the visual analogue scale(VAS) score was ≥ 4. The outcomes measured over 48 h after the operation were VAS scores both at rest and during mobilization, total morphine consumption, relative side effects, and basic vital signs. Further results including time to extubation, recovery of bowel function, surgical wound healing,mean length of hospitalization after surgery, and the patient's satisfaction were also recorded.RESULTS: All three groups had similar VAS scores during the first 48 h after surgery. Group CWI and group EA, compared with group PCIA, had lower morphine consumption(P 0.001), less postoperative nausea and vomiting(1.20 ± 0.41 vs 1.96 ± 0.67, 1.32 ± 0.56 vs 1.96 ± 0.67, respectively, P 0.001), earlier extubation(16.56 ± 5.24 min vs 19.76 ± 5.75 min, P 0.05, 15.48 ± 4.59 min vs 19.76 ± 5.75 min, P 0.01), and earlier recovery of bowel function(2.96 ± 1.17 d vs 3.60 ± 1.04 d, 2.80 ± 1.38 d vs 3.60 ± 1.04 d, respectively, P 0.05). The mean length of hospitalization after surgery was reduced in groups CWI(8.20 ± 2.58 d vs 10.08 ± 3.15 d, P 0.05) and EA(7.96 ± 2.30 d vs 10.08 ± 3.15 d, P 0.01) compared with group PCIA. All three groups had similar patient satisfaction and wound healing, but group PCIA was prone to higher sedation scores when compared with groups CWI and EA, especially during the first 12 h after surgery. Group EA had a lower mean arterial pressure within the first postoperative 12 h compared with the other two groups.CONCLUSION : CWI with ropivacaine yields a satisfactory analgesic effect within the first 48 h after open gastrectomy, with lower morphine consumption and accelerated recovery.     

Interplay of neuropilin-1 and semaphorin 3A after partial hepatectomy in rats

AIM:To elucidate the role of neuropilin-1(Nrp-1) and semaphorin 3A(Sema3A) in sinusoidal remodeling during liver regeneration in rats.METHODS:Male Wistar/ST rats at 7 wk of age,weighing about 200 g,were used for all animal experiments.In vivo,at 24,48,72,96,144 and 192 h after twothirds partial hepatectomy(PHx),the remnant livers were removed.Liver tissues were immunohistochemically stained for Nrp-1,Sema3A and SE-1,a liver sinusoidal endothelial cell(SEC) marker.Total RNA of the liver tissue was extracted and reversely transcribed into cDNA.The mRNA expression of Sema3A was analyzed by quantitative real-time polymerase chain reaction and normalized to that of ribosomal protein S18.In vitro,SECs were isolated from rat liver and cultured in endothelial growth medium containing 20 ng/mL vascular endothelial cell growth factor.Migration of SECs in primary culture was assessed by cell transwell assay with or without recombinant Sema3A.Apoptotic cells were determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method.RESULTS:In vitro,immunohistochemistry study revealed that Sema3A and Nrp-1 were constitutively expressed in hepatocytes and SECs,respectively,in normal rat liver tissues.Nrp-1 expression in SECs was quantified by the percentage of immunostained area with antiNrp-1 antibody in relation to the area stained with SE-1.Between 24 h and 96 h following resection of liver,Nrp-1 expression in SECs was transiently increased.Compared with the baseline(5.2% ± 0.1%),Nrp-1 expression in SECs significantly increased at 24 h(17.3% ± 0.7%,P 0.05),48 h(39.1% ± 0.6%,P 0.01),72 h(46.9% ± 4.5%,P 0.01) and 96 h(29.9% ± 3.8%,P 0.01) after PHx,then returned to the basal level at termination of liver regeneration.Interestingly,the expression of Sema3A was inversely associated with that of Nrp-1 in liver after PHx.Sema3A mRNA expression was significantly reduced by about 75% over the period 24-144 h after PHx(P 0.05),and returned to basal levels at 192 h after PHx.In vitro,SECs isolated from rats after PHx(PHx-SECs) were observed to migrate to the lower chamber of the cell transwell system after incubation for 24 h,but not cells from normal rats(CONT-SECs),indicating that mobility of PHx-SECs increases as compared with that of CONT-SECs.Moreover,recombinant Sema3A significantly attenuated migration in PHx-SECs in primary culture(vehicle-treated 100% ± 7.9% vs Sema3A-treated 42.6% ± 5.4%,P 0.01),but not in CONT-SECs.Compared with CONTSECs,the apoptotic rate of PHx-SECs decreased by 78.3%(P 0.05).There was no difference in apoptosis between CONT-SECs that were treated with vehicle and Sema3A.However,in PHx-SECs,apoptosis was induced by the presence of 5 nmol Sema3A for 24 h(vehicle-treated 21.7% ± 7.6% vs Sema3A-treated 104.3% ± 8.9%,P 0.05).In addition,immunohistochemistry confirmed the increased expression of Nrp-1 in PHx-SECs,while it was noted to a lesser extent in CONT-SECs.CONCLUSION:The interplay of Nrp-1 and Sema3A shown in our results may lead to a better understanding of interaction between sinusoidal remodeling and SECs during liver regeneration.     

Hepatocyte nuclear factor 4α induces a tendency of differentiation and activation of rat hepatic stellate cells

AIM: To investigate the effect of hepatocyte nuclear factor 4α(HNF4α) on the differentiation and transformation of hepatic stellate cells(HSCs).METHODS: By constructing the recombinant adenovirus vector expressing HNF4α and HNF4αshRNA vector, and manipulating HNF4α expression in HSC-T6 cells, we explored the influence of HNF4α and its induction capacity in the differentiation of rat HSCs into hepatocytes.RESULTS: With increased expression of HNF4αmediated by AdHNF4α, the relative expression of Nanog was downregulated in HSC-T6 cells(98.33 ±12.33 vs 41.33 ± 5.67, P 0.001). Consequently, the expression of G-P-6 and PEPCK was upregulated(G-P-6:14.34 ± 3.33 vs 42.53 ± 5.87, P 0.01; PEPCK: 10.10± 4.67 vs 56.56 ± 5.25, P 0.001), the expression of AFP and ALB was positive, and the expression of Nanog, Type Ⅰ collagen, α-SMA, and TIMP-1 was significantly decreased. HNF4α also downregulated vimentin expression and enhanced E-cadherin expression. The ultrastructure of HNF4α-induced cells had more mitochondria and ribosomes compared with the parental cells. After silencing HNF4α expression,EPCK, E-cadherin, AFP, and ALB were downregulated and α-SMA and vimentin were upregulated.CONCLUSION: HNF4α can induce a tendency of differentiation of HSCs into hepatocyte-like cells. These findings may provide an effective way for the treatmentof liver diseases.     

Ethyl pyruvate protects against experimental acute-on-chronic liver failure in rats

AIM:To investigate the protective effects of ethyl pyruvate(EP) on acute-on-chronic liver failure(ACLF) in rats.METHODS:An ACLF model was established in rats,and animals were randomly divided into normal,model and EP treatment groups.The rats in EP treatment group received EP(40 mg/kg) at 3 h,6 h,12 h and 24 h after induction of ACLF.Serum endotoxin,high mobility group box-1(HMGB1),alanine transaminase(ALT),tumor necrosis factor-(TNF-),interferon-(IFN-),interleukin(IL)-10 and IL-18 levels,changes of liver histology and HMGB1 expressions in liver tissues were detected at 48 h after induction of ACLF.The effects of EP on the survival of ACLF rats were also observed.RESULTS:Serum levels of endotoxin(0.394 ± 0.066 EU/mL vs 0.086 ± 0.017 EU/mL,P 0.001),HMGB1(35.42 ± 10.86 g/L vs 2.14 ± 0.27 g/L,P 0.001),ALT(8415.87 ± 3567.54 IU/L vs 38.64 ± 8.82 IU/L,P 0.001),TNF-(190.77 ± 12.34 ng/L vs 124.40 ± 4.12 ng/L,P 0.001),IFN-(715.38 ± 86.03 ng/L vs 398.66 ± 32.91 ng/L,P 0.001),IL-10(6.85 ± 0.64 ng/L vs 3.49 ± 0.24 ng/L,P 0.001) and IL-18(85.19 ± 3.49 ng/L vs 55.38 ± 1.25 ng/L,P 0.001) were significantly increased,and liver tissues presented severe pathological injury in the model group compared with the normal group.However,EP administration significantly improved hepatic histopathology and reduced the serum levels of endotoxin(0.155 ± 0.045 EU/mL vs 0.394 ± 0.066 EU/mL,P 0.001) and inflammatory cytokines(11.13 ± 2.58 g/L vs 35.42 ± 10.86 g/L for HMGB1,3512.86 ± 972.67 IU/L vs 8415.87 ± 3567.54 IU/L for ALT,128.55 ± 5.76 ng/L vs 190.77 ± 12.34 ng/L for TNF-,438.16 ± 38.10 ng/L vs 715.38 ± 86.03 ng/L for IFN-,3.55 ± 0.36 ng/L vs 6.85 ± 0.64 ng/L for IL-10,and 60.35 ± 1.63 ng/L vs 85.19 ± 3.49 ng/L for IL-18,respectively,P 0.001),and the levels of HMGB1 in liver tissues regardless of treatment time after induction of ACLF.EP treatment at the four time points prolonged the median survival time of ACLF rats(60 h) to 162 h,120 h,102 h and 78 h,respectively(2 = 41.17,P 0.0001).CONCLUSION:EP administration can protect against ACLF in rats,and is a potential and novel therapeutic agent for severe liver injury.     

Effect of Lianshu preparation on lipopolysaccharide-induced diarrhea in rats

AIM： To investigate the effect of Lianshu preparation on lipopolysaccharide （LPS）-induced diarrhea in rats. METHODS： A diarrhea model was established in Sprague Dawley rats via injection of 1 mL of 30 mg/kg LPS. A total of 40 rats were randomly divided into normal group, LPS group, LPS ＋ Lianshu group, LPS ＋ berberine group （n = 10 in each group）. Their intestinal mucosal barrier and frequency of diarrhea were observed. Levels of glucose, serum Na^＋, K^＋, Cl and hematocrit, plasma nitrogen monoxide （NO）, diamine oxidase （DAO）, and D （-）-lactate were measured. The number of IgA＋ plasma cells in small intestine was detected and SIgA levels in the intestinal fluid were measured. The antipyretic activity of Lianshu preparation in rats was evaluated using Brewer＇s yeast-induced pyrexia （10 mL/kg of 20% aqueous suspension）. Acetaminophen （250 mg/kg, intragastric administration, bid） was comparison. Temperature used as a standard drug for was recorded 1 h before and 6 h after Brewer＇s yeast injection. Finally, small intestina transmission in mice treated with Lianshu was detected after intraperitoneal injection of methyl prostigmin （2 mg/kg）. Atropine （10 g/kg） was used as a control. The ink content in intestine was determined and the total length of intestine was measured. RESULTS： The frequency of diarrhea was higher in LPS group than in LPS ＋ Lianshu group and LPS ＋ berberine group （36.70± 5.23 vs 28.50 ±4.06 and 32.70±9.30 respectively, P 〈 0.01）, and lower in LP5 ＋ Lianshu group than in LPS ＋ berberine group （P = 0.03）. The levels of Na＋, glucose, Cl, K^＋ were significantly lower in LPS ＋ Lianshu group than in LPS ＋ berberine group （140.35±3.19 mmol/L vs 131.99±4.86 mmol/L, 8.49 ±1.84 mmol/L vs 6.54±2.30 mmol/L, 106.29± 4.41 mmol/L vs 102.5±1.39 mmol/L, 5.08±0.66 mmol/L vs 4.32 ± 0.62 mmol/L respectively, P 〈 0.05）. The level of hematocrit was lower in LPS ＋ Lianshu group than in LPS ＋ berberine group （0.50% ±0.07% vs 0.59%± 0.10% respectively, P 〈 0.05）. The plasma levels of NO, DAO and D （-）-lactate were higher in LPS group than in normal group （79.74 ± 7.39μmol/L vs 24.94 ± 3.38μmol/L, 2.48 ±0.42μ/mL vs 0.82 ±0.33 p/mL, 5.63± 0.85μg/mL vs 2.01 ±0.32 μg/mL respectively, P 〈 0.01）, and lower in LPS ＋ Lianshu group than in LP5 ＋ berberine group （48.59±4.70μmol/L vs 51.56 ±8.38 μmol/L, 1.43± 0.53μmol/mL vs 1.81 ±0.42 μmol/mL, 4.00± 0.54 μg/mL vs 4.88 ± 0.77 pg/mL respectively, P 〈 0.05）. The morphology of the intestinal mucosa showed destroyed villi in LPS group and atrophied intestinal mucosa in other groups. The pathological intestinal mucosal changes were less in LPS ＋ Lianshu group than in LPS group. The number of IgA＋ plasma cells and amount of SIgA were higher in LPS ＋ Lianshu group than in LPS group （1.16±0.19/μm^2 vs 1.09±0.28/μm^2, P = 0.026; 0.59 ±0.12 mg/L vs 0.15± 0.19 mg/L respectively, P = 0.000）. Lianshu had counteractive effects on yeast-induced pyrexia and enterokinesia in rats. CONCLUSION： Lianshu preparation has therapeutic effects on LPS-induced diarrhea and enterokinesia in rats.     

Expression profile of polyunsaturated fatty acids in colorectal cancer

AIM:To investigate the relationship between the metabolism of polyunsaturated fatty acids(PUFAs)andtumor-associated factors for predicting the outcome of colorectal carcinoma(CRC)in Chinese patients.METHODS:Fresh-frozen malignant and normal tissues from 82 Chinese patients with CRC were analyzed for PUFA composition using gas-liquid chromatography.The levels of vascular endothelial growth factor(VEGF),cyclooxygenase-2(COX-2),prostaglandin E2 and platelet-derived growth factor(PDGF)were measured by enzyme-linked immunosorbent assay,and the levels of VEGF,p53 and Ki-67 were measured by immunohistochemistry.RESULTS:In malignant tissue,compared with normal tissue,the levels of totalω-6 PUFAs(24.64%±3.41%vs 26.77%±3.37%,P=0.00)and linoleic acid(LA)(15.46%±3.51%vs 18.30%±2.83%,P0.01)were lower,whereas the levels of totalω-3 PUFAs(1.58%±0.74%vs 1.35%±0.60%,P0.01)and dihomo-gamma-linolenic acid(DGLA)(1.32%±0.69%vs 0.85%±0.29%,P0.01)were significantly higher.The ratios of arachidonic acid(AA)/LA(0.53±0.22 vs0.42±0.19,P0.01)and AA/totalω-6 PUFAs(0.31±0.09 vs 0.27±0.10,P0.01)were also significantly higher in malignant tissue.The levels of PDGF(353.10±148.85 pg/m L vs 286.09±104.91 pg/m L,P0.01),COX-2(125.21±70.29 ng/m L vs 67.06±42.22 ng/m L,P0.01)and VEGF(357.11±128.76 pg/m L vs211.38±99.47 pg/m L,P0.01)were also higher in malignant tissue compared to normal tissue.COX-2was inversely correlated with LA(R=-0.3244,P0.05)and positively correlated with AA/totalω-6 PUFAs(R=0.3083,P0.05)and AA/LA(R=0.3001,P0.05).The tissue level of LA was highest in poorly differentiated tumors(19.9%±6.3%,P0.05),while the ratio of AA/ω-3 PUFAs was lowest in these tumors(10.8±2.6,P0.05).In VEGF-positive tumors,the level of LA was higher(16.2%±3.7%vs 13.9%±2.7%,P0.01),while the AA/ω-3PUFA,AA/ω-6 PUFA,and AA/LA ratios were lower than in VEGF-negativetumors(5.0±1.8 vs 6.7±3.3,0.30±0.09 vs 0.34±0.09,0.50±0.21 vs 0.61±0.21,P0.01).CONCLUSION:The metabolism of PUFAs may playan important role in the evolution of inflammationdriven tumorigenesis in CRC and may be considered apotential marker for prognosis.     

Three-dimensional vs two-dimensional video assisted thoracoscopic esophagectomy for patients with esophageal cancer

AIM: To define the benefits of three-dimensional video-assisted thoracoscopic esophagectomy(3D-VATE)over 2D-VATE for esophageal cancer.METHODS: A total of 93 patients with esophageal cancer including 45 patients receiving 3D-VATE and48 receiving 2D-VATE were evaluated. Data related to patient and cancer characteristics, operating time,intraoperative bleeding, morbidity and mortality,postoperative inflammatory markers, Numerical Rating Scale for postoperative pain, Constant-Murley rating system for shoulder recovery and oxygenation index(OI) were collected. All medical records were retrieved from a prospectively maintained oncological database at our institution. A retrospective study was performed to compare the short-term surgical outcomes between the two groups.RESULTS: No significant differences were found between the two groups in either morbidity or mortality(P = 0.328). An enhanced surgical recovery was noted in the 3D group as indicated by shortened thoracoscopic operation time(3D vs 2D: 68 ± 13.79 min vs 83 ± 13min, P 0.01), minor intraoperative blood loss(3D vs 2D: 68.2 ± 10.7 ml vs 89.8 ± 10.4 ml, P 0.01),earlier chest tube removal(3D vs 2D: 2.67 ± 1.01 vs3.75 ± 1.15 d, P 0.01), shorter length of hospital stay(3D vs 2D: 9.07 ± 2.00 vs 10.85 ± 3.40 d, P 0.01), lower in-hospital expenses(3D vs 2D: 74968.4± 9637.8 vs 86211.1 ± 8519.7 RMB, P 0.01), lower pain intensity(P 0.01) and faster recovery of the left shoulder function(P 0.01). Better preservation of the pulmonary function was also found in the 3D group as the decline of the OI post operation was significantly lower than that of the 2D group(P 0.01). Changes of postoperative inflammatory markers, including procalcitonin [postoperative days(PODs) 4 and 7: P 0.01], peripheral granulocytes(PODs 1, 4 and 7: P 0.01) and hypersensitive C-reactive protein(POD 4: P 0.01) in 3D-VATE patients were less than those in the 2D group. Moreover, utilization of the 3D technique extended the dissection of the thoracic lymph nodes(P 0.01), with better exposure of nodes in the left recurrent laryngeal nerve(P = 0.031).CONCLUSION: 3D-VATE could be a more viable technique over 2D-VATE in terms of short-term outcomes for patients with esophageal cancer.     

CD74 and macrophage migration inhibitory factor as therapeutic targets in gastric cancer

AIM: To investigate the relationship and molecular features of CD74/macrophage migration inhibitory factor (MIF)/Toll-like receptor 4 (TLR4) in gastric cancer.METHODS: CD74, MIF and TLR4 expression in the paraffin-embedded sections of gastric cancer from 120 patients were detected by immunohistochemical staining. Knock down of CD74 expression in gastric cancer cell line MKN-45 was performed by lentivirus transduction and detected by Western blotting. MKN-45 cell proliferation assay under the stimulants was measured by the cell counting kit 8 (CCK8) assay and MIF concentration in the culture medium was detected by enzyme-linked immunosorbent assay. Surface staining of CD74 in the MKN-45 cell line under the stimulation of lipopolysaccharide (LPS) was measured by flow cytometry. MIF, CD74 and TLR4 co-localization in the MKN-45 cell line was performed by the immunoprecipitation.RESULTS: CD74, MIF and TLR4 were found to be expressed in gastric cancer and increased significantly in the advanced stage, and were also associated with lymph node metastasis. Correlation analysis revealed that CD74 was positively correlated with MIF (r = 0.2367, P < 0.01) and both proteins were also associated with TLR4 (r = 0.4414, r = 0.5001, respectively, P < 0.01). LPS can significantly promote MKN-45 cell proliferation (3.027 ± 0.388 vs 4.201 ± 0.092, P < 0.05), induce MIF production (54.333 ± 2.906 pg/mL vs 29.667 ± 3.180 pg/mL, P < 0.01) and cell surface expression of CD74 (75.6% ± 4.046% vs 9.4% ± 0.964%, P < 0.01) at LPS concentration of 1 μg/mL compared to medium control. Knockdown of CD74 or using anti-CD74 and MIF antagonist ISO-1 significantly reduced LPS-induced MKN-45 cell proliferation (4.201 ± 0.092 vs 3.337 ± 0.087, 4.534 ± 0.222 vs 3.368 ± 0.290, 4.058 ± 0.292 vs 2.934 ± 0.197, respectively, P < 0.01). MIF, CD74 and TLR4 could co-localize in the MKN-45 cell line.CONCLUSION: Upregulation of MIF, CD74 and TLR4 are associated with increasing clinical stage and provide an opportunity as novel gastric cancer chemoprevention and/or treatment strategy.     

Are gastric mucosal macrophages responsible for gastric injury in acute pancreatitis?

AIM:To investigate the protective effect of clodronatecontaining liposomes against severe acute pancreatitis(SAP)-triggered acute gastric mucosal injury(AGMI) in rats.METHODS:Clodronate- and phosphate-buffered saline(PBS)-containing liposomes were prepared by reverse-phase evaporation.The SAP rat model was established by injecting sodium taurocholate into the pancreatic subcapsular space.Sprague-Dawley rats were randomly divided into three groups:control(C),SAP plus PBS-containing liposome(P) and SAP plus clodronate-containing liposome(T).Serum tumor necrosis factor(TNF)-α levels were estimated by ELISA.Pathological changes in the gastric mucosa and pancreas were observed by hematoxylin and eosin(HE) staining.Apoptotic cells were detected by terminal deoxynucleotidyl transferase d UTP nick end labeling staining.The numbers of macrophages in the gastric mucosa were analyzed by CD68 immunohistochemical staining.RESULTS:The liposomes had a mean diameter of 150 ± 30 nm.The TNF-α levels were significantly higher in the P group than that in the C group(2 h,145.13 ± 11.50 vs 23.2 ± 2.03; 6 h,245.06 ± 12.11 vs 30.28 ± 6.07,P < 0.05),and they were significantly lower in the T group than that in the P group(2 h,93.24 ± 23.11 vs 145.13 ± 11.50; 6 h,135.18 ± 13.10 vs 245.06 ± 12.11,P < 0.05).The pathological scores of the pancreas were lower in the T group than in the P group(2 h,1.88 ± 0.83 vs 4.13 ± 0.83; 6 h,2.87 ± 0.64 vs 6.25 ± 0.88,P < 0.01).The pathological scores of the gastric mucosa were also lower in the T group than in the P group(2 h,1.12 ± 0.64 vs 2 ± 0.75; 6 h,1.58 ± 0.53 vs 3 ± 1.31,P < 0.05).In addition,increased CD68 levels were observed in the gastric mucosa of the P group compared with the C group.Clodronate-containing liposomes decreased the CD68 levels in the mucosa of the T group.The apoptotic indexes of the gastric mucosa were higher in the T group than in the P group(2 h,15.7 ± 0.92 vs 11.5 ± 1.64; 6 h,21.12 ± 1.06 vs 12.6 ± 2.44,P < 0.01).CONCLUSION:Gastric macrophages contribute to the pathogenesis of gastric injury in SAP.Clodronatecontaining liposomes have protective effects against AGMI in rats with SAP.