Moxibustion inhibits interleukin-12 and tumor necrosis factor alpha and modulates intestinal flora in rat with ulcerative colitis

AIM: To investigate the effect of moxibustion on intestinal flora and release of interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α) from the colon in rat with ulcerative colitis (UC).METHODS: A rat model of UC was established by local stimulation of the intestine with supernatant from colonic contents harvested from human UC patients. A total of 40 male Sprague-Dawley rats were randomly divided into the following groups: normal (sham), model (UC), herb-partition moxibustion (HPM-treated), and positive control sulfasalazine (SA-treated). Rats treated with HPM received HPM at acupuncture points ST25 and RN6, once a day for 15 min, for a total of 8 d. Rats in the SA group were perfused with SA twice a day for 8 d. The colonic histopathology was observed by hematoxylin-eosin. The levels of intestinal flora, including Bifidobacterium, Lactobacillus, Escherichia coli (E. coli), and Bacteroides fragilis (B. fragilis), were tested by real-time quantitative polymerase chain reaction to detect bacterial 16S rRNA/DNA in order to determine DNA copy numbers of each specific species. Immunohistochemical assays were used to observe the expression of TNF-α and IL-12 in the rat colons.RESULTS: HPM treatment inhibited immunopathology in colonic tissues of UC rats; the general morphological score and the immunopathological score were significantly decreased in the HPM and SA groups compared with the model group [3.5 (2.0-4.0), 3.0 (1.5-3.5) vs 6.0 (5.5-7.0), P < 0.05 for the general morphological score, and 3.00 (2.00-3.50), 3.00 (2.50-3.50) vs 5.00 (4.50-5.50), P < 0.01 for the immunopathological score]. As measured by DNA copy number, we found that Bifidobacterium and Lactobacillus, which are associated with a healthy colon, were significantly higher in the HPM and SA groups than in the model group (1.395 ± 1.339, 1.461 ± 1.152 vs 0.045 ± 0.036, P < 0.01 for Bifidobacterium, and 0.395 ± 0.325, 0.851 ± 0.651 vs 0.0015 ± 0.0014, P < 0.01 for Lactobacillus). On the other hand, E. coli and B. fragilis, which are associated with an inflamed colon, were significantly lower in the HPM and SA groups than in the model group (0.244 ± 0.107, 0.628 ± 0.257 vs 1.691 ± 0.683, P < 0.01 for E. coli, and 0.351 ± 0.181, 0.416 ± 0.329 vs 1.285 ± 1.039, P < 0.01 for B. fragilis). The expression of TNF-α and IL-12 was decreased after HPM and SA treatment as compared to UC model alone (4970.81 ± 959.78, 6635.45 ± 1135.16 vs 12333.81 ± 680.79, P < 0.01 for TNF-α, and 5528.75 ± 1245.72, 7477.38 ± 1259.16 vs 12550.29 ± 1973.30, P < 0.01 for IL-12).CONCLUSION: HPM treatment can regulate intestinal flora and inhibit the expression of TNF-α and IL-12 in the colon tissues of UC rats, indicating that HPM can improve colonic immune response.     

Gegen Qinlian decoction enhances immunity and protects intestinal barrier function in colorectal cancer patients via gut microbiota

BACKGROUNDWe previously showed, using the Traditional Chinese Medicine System Pharmacology Database, that Gegen Qinlian decoction (GQD) had a direct antitumor effect, and was combined with programmed cell death protein (PD)-1 inhibitors to treat microsatellite stable (MSS) tumor-bearing mice. However, the effect of GQD on patients with colorectal cancer (CRC) is not clear.AIMTo determine the therapeutic mechanism of GQD in improving immune function, reducing inflammation and protecting intestinal barrier function.METHODSSeventy patients with CRC were included in this study: 37 in the control group and 33 in the treatment group. The proportions of CD4⁺ T, CD8⁺ T, natural killer (NK), NKT and T regulatory cells were measured by flow cytometry. Levels of the cytokines tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, IL-6, IL-10 and serotonin (5-hydroxytryptamine; 5-HT) in serum were assessed by enzyme-linked immunosorbent assay (ELISA). The expression of zonula occludens (ZO)-1, occludin, nuclear factor (NF)-κB and TNF-α in tumor and normal tissues was measured by immunohistochemistry. The composition of gut microbiota from patients in the treatment group was assessed using 16S rDNA analysis. RESULTSThere were no adverse events in the treatment group. The proportion of CD4⁺ T cells and NKT cells in the post-treatment group was significantly higher than that in the pre-treatment and control groups (P < 0.05). The level of TNF-α in the post-treatment group was significantly lower than that in the pre-treatment and control groups (P < 0.05). The concentration of 5-HT in the post-treatment group was significantly lower than that in the pre-treatment group (P < 0.05). The expression of ZO-1 and occludin in tumor tissues in the treatment group was significantly higher than that in the control group (P < 0.05). The expression of ZO-1 in normal tissues of the treatment group was significantly higher than that in the control group (P = 0.010). Compared with the control group, expression of NF-κB and TNF-α in tumor tissues of the treatment group was significantly decreased (P < 0.05). Compared with the pre-treatment group, GQD decreased the relative abundance of Megamonas and Veillonella. In addition, GQD increased the relative abundance of Bacteroides, Akkermansia and Prevotella. CONCLUSIONGQD enhances immunity and protects intestinal barrier function in patients with CRC by regulating the composition of gut microbiota.     

Moxibustion combined with acupuncture increases tight junction protein expression in Crohn's disease patients

AIM:To investigate the effect of herb-partitioned moxibustion combined with acupuncture on the expression of intestinal epithelial tight junction(TJ) proteins.METHODS:Sixty patients diagnosed with mild to moderate Crohn’s disease(CD)were allocated into the herb-partitioned moxibustion combined with acupuncture(HMA)group(n=30)or the mesalazine(MESA)group(n=30)using a parallel control method.There were 2 sets of acupoints used alternately for HMA treatment.The following points were included in Set A:ST25(Tianshu),RN6(Qihai),and RN9(Shuifen)for herb-partitioned moxibustion and ST36(Zusanli),ST37(Shangjuxu),LI11(Quchi),and LI4(Hegu)for acupuncture.The points for Set B included BL23(Shenshu)and BL25(Dachangshu)for herb-partitioned moxibustion and EX-B2 of T6-T1(Jiajixue)fo r acupuncture.The patients received the same treatment6 times a week for 12 consecutive weeks.The MESA group received 1 g of mesalazine enteric coated tablets4 times daily for 12 consecutive weeks.Intestinaltissues were stained and examined to compare the morphological and ultrastructural changes before and after the treatment session.Immunohistochemistry and in situ hybridization assays were used to detect the expression of intestinal epithelial TJ proteins zonula occludens-1(ZO-1),occludin,and claudin-1.The m RNA levels were also evaluated.RESULTS:After the treatment,both herb-partitioned moxibustion combined with acupuncture and mesalazine improved intestinal morphology and ultrastructure of CD patients;the patients treated with HMA showed better improvement.HMA significantly increased the expression of ZO-1(P=0.000),occludin(P=0.021),and claudin-1(P=0.016).MESA significantly increased the expression of ZO-1(P=0.016)and occludin(P=0.026).However,there was no significant increase in the expression of claudin-1(P=0.935).There was no statistically significant difference between the two groups for the expression of occludin and claudin-1(P0.05).The HMA group showed a significant improvement in ZO-1 expression compared to the MESA group(2333.34±352.51 vs 2160.38±307.08,P=0.047).HMA significantly increased the expression of ZO-1 m RNA(P=0.000),occludin m RNA(P=0.017),and claudin-1 m RNA(P=0.017).MESA significantly increased the expression of ZO-1 m RNA(P=0.000),occludin m RNA(P=0.042),and claudin-1 m RNA(P=0.041).There was no statistically significant difference between the two groups in the expression of occludin and claudin-1 m RNA(P0.05).However,the HMA group showed a significant improvement in ZO-1 m RNA expression compared with the MESA group(2378.17±308.77 vs 2200.56±281.88,P=0.023).CONCLUSION:HMA can repair intestinal epithelial barrier lesions and relieve inflammation by upregulating the expression of TJ proteins and their m RNAs.     

Nimbolide inhibits tumor growth by restoring hepatic tight junction protein expression and reduced inflammation in an experimental hepatocarcinogenesis

BACKGROUNDAltered tight junction (TJ) proteins are correlated with carcinogenesis and tumor development. Nimbolide is a tetranotriterpenoid that has been shown to have antioxidant and anti-proliferative properties; however, its anticancer effects and molecular mechanism in hepatocellular carcinoma (HCC) remains obscure.AIMTo investigate the effect of nimbolide on TJ proteins, cell cycle progression, and hepatic inflammation in a mouse model of HCC. METHODSHCC was induced in male Swiss albino mice (CD-1 strain) by a single intraperitoneal injection of 100 mg/kg diethylnitrosamine (DEN) followed by 80 ppm N-nitrosomorpholine (NMOR) in drinking water for 28 wk. After 28 wk, nimbolide (6 mg/kg) was given orally for four consecutive weeks in DEN/NMOR induced HCC mice. At the end of the 32^nd week, all the mice were sacrificed and blood and liver samples were collected for various analyses. Macroscopic examinations of hepatic nodules were assessed. Liver histology and HCC tumor markers such as alpha-fetoprotein (AFP) and glypican-3 were measured. Expression of TJ proteins, cell proliferation, and cell cycle markers, inflammatory markers, and oxidative stress markers were analyzed. In silico analysis was performed to confirm the binding and modulatory effect of nimbolide on zonula occludens 1 (ZO-1), nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB), and tumor necrosis factor alpha (TNF-α).RESULTSWe found nimbolide treatment at a concentration of 6 mg/kg to HCC mice reduced hepatic tumor size by 52.08% and tumor volume (P < 0.01), and delayed tumor growth in HCC mice with a concomitant reduction in tumor markers such as AFP levels (P < 0.01) and glypican-3 expression (P < 0.05). Furthermore, nimbolide treatment increased tight junction proteins such as ZO-1 and occludin expression (P < 0.05, respectively) and reduced ZO-1 associated nucleic acid binding protein expression (P < 0.001) in HCC mice liver. Nimbolide treatment to HCC mice also inhibited cell proliferation and suppressed cell cycle progression by attenuating proliferating cell nuclear antigen (P < 0.01), cyclin dependent kinase (P < 0.05), and CyclinD1 (P < 0.05) expression. In addition, nimbolide treatment to HCC mice ameliorated hepatic inflammation by reducing NF-κB, interleukin 1 beta and TNF-α expression (P < 0.05, respectively) and abrogated oxidative stress by attenuating 4-hydroxynonenal expression (P < 0.01). Molecular docking studies further confirmed that nimbolide interacts with ZO-1, NF-κB, and TNF-α.CONCLUSIONOur current study showed for the first time that nimbolide exhibits anticancer effect by reducing tumor size, tumor burden and by suppressing cell cycle progression in HCC mice. Furthermore, nimbolide treatment to HCC mice ameliorated inflammation and oxidative stress, and improved TJ proteins expression. Consequently, nimbolide could be potentially used as a natural therapeutic agent for HCC treatment, however further human studies are warranted.     

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

AIM: To investigate the effects of moxibustion on down-regulation of the colonic epithelial cell apoptosis and repair of the tight junctions in rats with Crohn’s disease (CD). METHODS: Sixty male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an herbs-partitioned moxibustion (HPM) group, a mild-warm moxibustion (MWM) group and a salicylazosulphapyridine (SASP) group, with 12 rats in each group. The CD model rats were treated with trinitrobenzene sulph...     

p53 antibodies, metallothioneins, and oxidative stress markers in chronic ulcerative colitis with dysplasia

AIM:To investigate the role of p53 antibodies (p53Abs),metallothioneins (MTs) and oxidative stress markers in the early detection of dysplasia in chronic ulcerative colitis (UC).METHODS:The study included 30 UC patients,15 without dysplasia (group Ⅱ) and 15 with dysplasia (group Ⅲ),in addition to 15 healthy volunteers (group Ⅰ,control subjects).The enzyme-linked immunosorbent assay technique was used to measure serum p53Abs and MTs,while advanced oxidation protein products (AOPPs),and reduced glutathione (G...     

Protective effect of glutamine on intestinal injury and bacterial community in rats exposed to hypobaric hypoxia environment

AIM:To investigate the protective effect of glutamine(Gln)on intestinal injury and the bacterial community in rats exposed to hypobaric hypoxia environment.METHODS:Sprague-Dawley rats were divided into control,hypobaric hypoxia(HH),and hypobaric hypoxia+Gln(5.0 g/kg BW·d)(HG)groups.On the first 3 d,all rats were placed in a normal environment.After the third day,the HH and HG groups were transferred into a hypobaric chamber at a simulated elevation of 7000m for 5 d.The rats in the HG group were given Gln by gavage daily for 8 d.The rats in the control and HH groups were treated with the same volume of saline.The intestinal morphology,serum levels of malondialdehyde(MDA),superoxide dismutase(SOD),interleukin-6 (IL-6),tumor necrosis factor-α(TNF-α),interferon-gamma(IFN-γ)and diamino oxidase(DAO)were examined.We also evaluated the expression levels of occludin,toll-like receptor 4(TLR4),nuclear factor-κB p65(NF-κB p65)and myeloid differentiation factor 88(MyD88),and examined the bacterial community in caecal contents.RESULTS:Hypobaric hypoxia induced the enlargement of the heart,liver,lung and kidney,and caused spleen atrophy.Intestinal villi damage was also observed in the HH group.Supplementation with Gln significantly alleviated hypobaric-induced damage to main organs including the intestine,increased serum SOD(1.14±0.03 vs 0.88±0.04,P<0.05)and MDA(8.35±1.60,P<0.01)levels and decreased serum IL-6(1172.13±30.49 vs 1407.05±34.36,P<0.05),TNF-α(77.46±0.78 vs 123.70±3.03,P<0.001),IFN-γ(1355.42±72.80 vs 1830.16±42.07,P<0.01)and DAO(629.30±9.15 vs 524.10±13.34,P<0.001)levels.Moreover,Gln significantly increased occludin(0.72±0.05 vs 0.09±0.01,P<0.001),TLR4(0.15±0.05 vs 0.30±0.09,P<0.05),MyD88(0.32±0.08 vs 0.71±0.06,P<0.01),and NF-κB p65(0.16±0.04 vs 0.44±0.03,P<0.01)expression levels and improved the intestinal bacterial community.CONCLUSION:Gln treatment protects from intestinal injury and regulates the gut flora imbalance in hypoxia environment.These effects may be related to the TLR4/MyD88/NF-κB signaling pathway.     

Dimethyl sulfoxide inhibits zymosan-induced intestinal inflammation and barrier dysfunction

AIM: To investigate whether dimethyl sulfoxide(DMSO) inhibits gut inflammation and barrier dysfunction following zymosan-induced systemic inflammatoryresponse syndrome and multiple organ dysfunction syndrome.METHODS: Sprague-Dawley rats were randomly divided into four groups: sham with administration of normal saline(SS group); sham with administration of DMSO(SD group); zymosan with administration of normal saline(ZS group); and zymosan with administration of DMSO(ZD group). Each group contained three subgroups according to 4 h,8 h,and 24 h after surgery. At 4 h,8 h,and 24 h after intraperitoneal injection of zymosan(750 mg/kg),the levels of intestinal inflammatory cytokines [tumor necrosis factor-alpha(TNF-α) and interleukin(IL)-10] and oxides(myeloperoxidase,malonaldehyde,and superoxide dismutase) were examined. The levels of diamine oxidase(DAO) in plasma and intestinal mucosal blood flow(IMBF) were determined. Intestinal injury was also evaluated using an intestinal histological score and apoptosis of intestinal epithelial cells was determined by deoxynucleotidyl transferase d UTP nick end labeling(TUNEL) assay. The intestinal epithelial tight junction protein,ZO-1,was observed by immunofluorescence.RESULTS: DMSO decreased TNF-α and increased IL-10 levels in the intestine compared with the ZS group at the corresponding time points. The activity of intestinal myeloperoxidase in the ZS group was higher than that in the ZD group 24 h after zymosan administration(P 0.05). DMSO decreased the content of malondialdehyde(MDA) and increased the activity of superoxide dehydrogenase(SOD) 24 h after zymosan administration. The IMBF was lowest at 24 h and was 49.34% and 58.26% in the ZS group and ZD group,respectively(P 0.05). DMSO alleviated injury in intestinal villi,and the gut injury score was significantly lower than the ZS group(3.6 ± 0.2 vs 4.2 ± 0.3,P 0.05). DMSO decreased the level of DAO in plasma compared with the ZS group(65.1 ± 4.7 U/L vs 81.1 ± 5.0 U/L,P 0.05). DMSO significantly preserved ZO-1 protein expression and localization 24 h after zymosan administration. The TUNEL analysis indicated that the number of apoptotic intestinal cells in the ZS group was much higher than the ZD group(P 0.05).CONCLUSION: DMSO inhibited intestinal cytokines and protected against zymosan-induced gut barrier dysfunction.     

Protective effect of naringenin on acetic acid-induced ulcerative colitis in rats

AIM:To evaluate the ameliorative effect of naringenin(NG)during ulcerative colitis(UC)in rats.METHODS:Rats were treated with three different doses(25,50 and 100 mg/kg per day)of NG and a single dose of mesalazine(MES,300 mg/kg per day)for seven days prior to ulcerative colitis induction by4%acetic acid(AA).Twenty four hours after AA rectal administration,animals were scarified and the colonic tissues were dissected.Colonic mucus content was estimated using Alcian blue dye binding technique.In colon tissues,levels of total glutathione sulphadryls(T-GSH),non-protein sulphadryls(NP-SH)and thiobarbituric acid reactive substances(TBARS)were evaluated.The activities of the antioxidant enzymes,catalase(CAT)and superoxide dismutase(SOD)were measured.Concentrations of nucleic acids(DNA and RNA)and total protein were also estimated in colon tissues.Colonic levels of tumor necrosis factor-(TNF-),interleukin-1(IL-1),interleukin-6(IL-6),prostaglandin E2(PGE2)and nitric oxide(NO)were estimated.In cross section of colitis tissue the histopathological changes were observed.RESULTS:Colonic mucus content was decreased in AA compared to controls(587.09±65.59 mg/kg vs941.78±68.41 mg/kg,P<0.001).AA administration markedly reduced T-GSH(5.25±0.37 nmol/L vs 3.04±0.24 nmol/L,P<0.01),NP-SH(3.16±0.04 nmol/L vs 2.16±0.30 nmol/L,P<0.01),CAT(6.77±0.40 U/mg vs 3.04±0.2 U/mg,P<0.01)and SOD(3.10±0.11U/mg vs 1.77±0.18 U/mg,P<0.01)while TBARS,TNF-,IL-1,IL-6,PGE2 and NO levels(15.09±3.84nmol/L vs 59.90±16.34 nmol/L,P<0.01;113.56±1.91 pg/mg vs 134.24±4.77 pg/mg,P<0.01;209.20±36.38 pg/mg vs 422.19±31.47 pg/mg,P<0.01;250.83±25.09 pg/mg vs 638.58±115.9 pg/mg,P<0.01;248.19±36.98 pg/mg vs 541.74±58.34 pg/mg,P<0.01 and 81.26±2.98 mmol/g vs 101.90±10.73 mmol/g,P<0.001)were increased in colon of rats with UC compared controls respectively.Naringenin supplementation,significantly and dose dependently increased the colonic mucus content.The elevated TBARS levels were significantly decreased(39.35±5.86n     

Tumor necrosis factor alpha increases intestinal permeability in mice with fulminant hepatic failure

AIM:To determine the effect of tumor necrosis factor alpha(TNF-α) on intestinal permeability(IP) in mice with fulminant hepatic failure(FHF),and the expression of tight junction proteins.METHODS:We selected D-lactate as an index of IP,induced FHF using D-galactosamine/lipopolysaccharide and D-galactosamine/TNF-α,assessed the results using an enzymatic-spectrophotometric method,transmission electron microscopy,immunohistochemistry,Western blotting and real-time quantitative polymerase chain reaction.The effect of the administration of antiTNF-α immunoglobulin G(IgG) antibody,before the administration of D-galactosamine/lipopolysaccharide,on TNF-α was also assessed.RESULTS:IP was significantly increased in the mouse model of FHF 6 h after injection(13.57 ± 1.70 mg/L,13.02 ± 1.97 mg/L vs 3.76 ± 0.67 mg/L,P = 0.001).Electron microscopic analysis revealed tight junction(TJ) disruptions,epithelial cell swelling,and atrophy of intestinal villi.Expression of occludin and claudin-1 mRNA was significantly decreased in both FHF models(occludin:0.57 ± 0.159 fold vs baseline,P = 0.000;claudin-1:0.3067 ± 0.1291 fold vs baseline,P = 0.003),as were the distribution density of proteins in the intestinal mucosa and the levels of occludin and claudin-1 protein(occludin:0.61 ± 0.0473 fold vs baseline,P = 0.000;claudin-1:0.6633 ± 0.0328 fold vs baseline,P = 0.000).Prophylactic treatment with antiTNF-α IgG antibody prevented changes in IP(4.50 ± 0.97 mg/L vs 3.76 ± 0.67 mg/L,P = 0.791),intestinal tissue ultrastructure,and the mRNA levels of occludin and claudin-1 expression(occludin:0.8865 ± 0.0274 fold vs baseline,P = 0.505;claudin-1:0.85 ± 0.1437 fold vs baseline,P = 0.1),and in the protein levels(occludin:0.9467 ± 0.0285 fold vs baseline,P 0.05;claudin-1:0.9533 ± 0.0186 fold vs baseline,P = 0.148).CONCLUSION:Increased in IP stemmed from the downregulation of the TJ proteins occludin and claudin-1,and destruction of the TJ in the colon,which were induced by TNF-α in FHF mice.     

Protective role of hydrogen-rich water on aspirin-induced gastric mucosal damage in rats

AIM: To investigate the role of the hydrogen-rich water(HRW) in the prevention of aspirin-induced gastric mucosal injury in rats. METHODS: Forty male rats were allocated into four groups: normal control group, HRW group, aspirin group, and HRW plus aspirin group. The protective efficacy was tested by determining the gastric mucosal damage score. Malondialdehyde(MDA), superoxide dismutase(SOD), myeloperoxidase(MPO), interleukin(IL)-06 and tumor necrosis factor(TNF)-α in gastric tissues were evaluated. The serum levels of IL-1β and TNF-α were also detected. Histopathology of gastric tissues and localization of Cyclooxygenase 2(COX-2) were detected using hematoxylin and eosin staining and immunohistochemistry, respectively. RESULTS: Pretreatment with HRW obviously reduced aspirin-induced gastric damage scores(4.04 ± 0.492 vs 2.10 ± 0.437, P < 0.05). The oxidative stress levels of MDA and MPO in the gastric tissues increased significantly in the aspirin-treated group compared with the HRW group(2.43 ± 0.145 vs 1.79 ± 0.116 nmol/mg prot, P < 0.05 and 2.53 ± 0.238 vs 1.40 ± 0.208 U/g tissue, P < 0.05, respectively). HRW could obviously elevated the SOD levels in the gastric tissues(37.94 ± 8.44 vs 59.55 ± 9.02 nmol/mg prot, P < 0.05). Pretreatment with HRW significantly reduced IL-06 and TNF-α in the gastric tissues(46.65 ± 5.50 vs 32.15 ± 4.83 pg/mg, P < 0.05 and 1305.08 ± 101.23 vs 855.96 ± 93.22 pg/mg, P < 0.05), and IL-1β and TNF-α in the serum(505.38 ± 32.97 vs 343.37 ± 25.09 pg/mL, P < 0.05 and 264.53 ± 28.63 vs 114.96 ± 21.79 pg/mL, P < 0.05) compared to treatment with aspirin alone. HRW could significantly decrease the COX-2 expression in the gastric tissues(staining score: 8.4 ± 2.1 vs 2.9 ± 1.5, P < 0.05). CONCLUSION: HRW pretreatment alleviated the aspirin-induced gastric lesions by inhibiting the oxidative stress, inflammatory reaction and reducing the COX-2 in the gastric tissues.     

I_κB kinase-beta inhibitor attenuates hepatic fibrosis in mice

AIM: To investigate the anti-fibrosis effect of IκB kinase-beta inhibitor (IKK2 inhibitor IMD0354) in liver fibrosis. METHODS: Twenty male C57BL6 mice were divided into four groups. Five high-fat fed mice were injected with lipopolysaccharide (LPS, 10 mg/kg) intraperitoneally and five high-fat fed mice were without LPS injection to build models of liver injury, and the intervention group (five mice) was injected intraperitoneally with IKK2 inhibitor (IMD 30 mg/kg for 14 d), while the remaining five mice rec...     

Role of moxibustion in inflammatory responses during treatment of rat ulcerative colitis

AIM: To investigate the efficacy of moxibustion in ulcerative colitis (UC) rats from morphological, immunological and molecular biological perspectives.METHODS: Thirty-two Sprague-Dawley rats were randomly assigned to a blank control group (normal rats, n = 6) and a model replication (MR) group (UC rats, n = 26). A UC model was established by 2,4,6-trinitrobenzenesulfonic acid/dextran sulfate sodium enema. Rats in the MR group were further randomly assigned to a 9-min moxibustion (9M) group (9 moxa-cone, n = 6), 6-min moxibustion (6M) group (6 moxa-cone, n = 6), 3-min moxibustion (3M) group (3 moxa-cone, n = 6), and a waiting list control (WLC) group (no moxibustion treatment, n = 6). Rats in the moxibustion treatment group were treated in 14 sessions over 28 d. Disease activity, local tissue morphology, serum level of interleukin (IL)-8 and IL-10, and expression of Toll-like receptor (TLR)9 as well as nuclear factor (NF)-κB p65 in colonic tissue were determined by disease activity index (DAI), hematoxylin and eosin staining, electron microscopy, enzyme-linked immunosorbent assay and Western blotting, respectively.RESULTS: DAI was lowest in the 9M group and highest in the WLC group. The differences in DAI between the moxibustion treatment (3M, 6M, 9M) and no treatment groups were significant for all one-to-one comparisons (0.60 ± 0.54 vs 1.20 ± 0.44, 0.60 ± 0.54 vs 1.80 ± 0.45, 0.60 ± 0.54 vs 3.0 ± 0.45, respectively, P < 0.05). Light and electron microscopy showed that the neatness of the glandular arrangement in colonic mucosal epithelia gradually increased in the WLC, 3M, 6M to 9M groups. IL-8 level successively decreased while IL-10 level increased from the WLC to 3M, 6M and 9M groups. The differences among these groups were significant for all comparisons (105.46 ± 8.75 vs 76.61 ± 3.58, 105.46 ± 8.75 vs 69.78 ± 1.87, 105.46 ± 8.75 vs 67.41 ± 1.84, respectively, P < 0.01 for IL-8; and 30.83 ± 1.29 vs 75.64 ± 1.90, 30.83 ± 1.29 vs 80.90 ± 3.16, 30.83 ± 1.29 vs 83.46 ± 2.37, respectively, P < 0.01 for IL-10), except comparison of 6M vs 9M. Expression of TLR9 and NF-κB p65 decreased in order: highest in the WLC group and lowest in the 9M group. In addition, the differences among the WLC, 3M, 6M and 9M groups were significant for all comparisons (0.492 ± 0.026 vs 0.380 ± 0.022, 0.492 ± 0.026 vs 0.355 ± 0.005, 0.492 ± 0.026 vs 0.327 ± 0.015, respectively, P < 0.05 for TLR9; and 0.436 ± 0.041 vs 0.326 ± 0.022, 0.436 ± 0.041 vs 0.293 ± 0.006, 0.436 ± 0.041 vs 0.265 ± 0.017, respectively, P < 0.05 for NF-κB p65).CONCLUSION: Moxibustion repairs damaged colonic mucosa, suppresses serum IL-8, activates serum IL-10 level, and decreases expression of TLR-9 and NF-κB p65 in UC rats.     

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     

Mechanism of action of gypenosides on type 2 diabetes and non-alcoholic fatty liver disease in rats

AIM:To explore the mechanism of action of gypenosides(GPs)on type 2 diabetes mellitus and non-alcoholic fatty liver disease(T2DM-NAFLD)in rats.METHODS:Sixty rats were randomly divided into a healthy group,an untreated disease model group andGP-treatment groups.The study involved the evaluation of biochemical parameters,including serum aspartate transaminase(AST),alanine transferase(ALT),blood glucose(BG),triglycerides(TG)and total cholesterol(TC).Additionally,the protective effect of the treatments were confirmed histopathologically and the expression of TNF-αand NF-κB in the rat liver was analyzed using immunohistochemistry.The expression of proliferatoractivated receptor gamma(PPARγ)and cytochrome P450(CYP450)1A1 m RNA was determined by quantitative RTPCR.RESULTS:GP treatments at oral doses of 200,400,and800 mg/kg per day significantly decreased the levels of serum AST and ALT(P0.05,P0.01),especially at the dose of 800 mg/kg per day.To a similar extent,GP at800 mg/kg per day reduced the levels of BG(4.19±0.47,P0.01),TG(80.08±10.05,P0.01),TC(134.38±16.39,P0.01)and serum insulin(42.01±5.04,P0.01).The expression of TNF-αand NF-κB measured by immunohistochemistry was significantly reduced by GPs in a dose-dependent manner,and the expression of PPARγand CYP4501A1 m RNA,as measured using quantitative real-time PCR,were significantly down-regulated by GPs.Moreover,GPs decreased the infiltration of liver fats and reversed the histopathological changes in a dosedependent manner.CONCLUSION:This study suggests that GPs have a protective effect against T2DM-NAFLD by down-regulating the expression of TNF-αand NF-κB proteins,and PPARγand CYP4501A1 m RNAs.     

Overexpressed miRNA-155 dysregulates intestinal epithelial apical junctional complex in severe acute pancreatitis

AIM:To investigate whether miRNA-155(miR-155)dysregulates apical junctional complex(AJC)protein expression in experimental severe acute pancreatitis(SAP).METHODS:Twenty-four male BALB/c mice were randomly assigned to two groups:the SAP group(n=12)receiving sequential intraperitoneal injection of 50μg/kg caerulein and 10 mg/kg lipopolysaccharide over 6h,and the control group(n=12)receiving intraperitoneal injection of normal saline.Animals were sacrificed3 h following the last injection for collection of blood samples and pancreas and distal ileal segment specimens.Routine pancreas and intestine histology was used to assess SAP pathology and intestinal epithelial barrier damage.Levels of serum amylase,diamine oxidase(DAO),and tumor necrosis factor(TNF)-αwere determined using commercial kits.Total RNA samples were isolated from intestinal epithelial specimens and reversely transcribed into cDNA.miR-155 and RhoA mRNA expression profiles were determined using quantitative real-time polymerase chain reaction.Target genes for miR-155 were predicted using the miRTarBase database,RNA22 and PicTar computational methods.Western blotting was performed to quantitate the protein expression levels of the target gene RhoA,as well as zonula occludens(ZO)-1 and E-cadherin,two AJC component proteins.RESULTS:Intraperitoneal injection of caerulein and lipopolysaccharide successfully induced experimental acute pancreatic damage(SAP vs control,10.0±2.0vs 3.2±1.2,P<0.01)and intestinal epithelial barrier damage(3.2±0.7 vs 1.4±0.7,P<0.01).Levels of serum amylase(21.6±5.1 U/mL vs 14.3±4.2 U/mL,P<0.01),DAO(21.4±4.1 mg/mL vs 2.6±0.8 mg/mL,P<0.01),and TNF-α(61.0±15.1 ng/mL vs 42.9±13.9 ng/mL,P<0.01)increased significantly in SAP mice compared to those in control mice.miR-155 was significantly overexpressed in SAP intestinal epithelia(1.94±0.50 fold vs 1.03±0.23 fold,P<0.01),and RhoA gene containing three miR-155-specific binding sites in the three prime untranslated regions was one of the target genes for mi     

Kefir treatment ameliorates dextran sulfate sodium-induced colitis in rats

AIM: To investigate the preventive effect of kefir on colitis induced with dextran sulfate sodium(DSS) in rats.METHODS: Twenty-four male Wistar-albino rats were randomized into four groups: normal control,kefircontrol,colitis,and kefir-colitis groups. Rats in the normal and kefir-control groups were administered tap water as drinking water for 14 d. Rats in the colitis and kefir-colitis groups were administered a 3% DSS solution as drinking water for 8-14 d to induce colitis. Rats in the kefir-control and kefir-colitis groups were administered 5 m L kefir once a day for 14 d while rats in the normal control and colitis group were administered an identical volume of the placebo(skim milk) using an orogastric feeding tube. Clinical colitis was evaluated with reference to the disease activity index(DAI),based on daily weight loss,stool consistency,and presence of bleeding in feces. Rats were sacrificed on the 15 th day,blood specimens were collected,and colon tissues were rapidly removed. Levels of myeloperoxidase(MPO),tumor necrosisfactor(TNF)-α,interleukin(IL)-10,malondialdehyde,and inducible nitric oxide synthase(i NOS) were measured in colon tissue.RESULTS: The DAI was lower in the kefir-colitis group than in the colitis group(on the 3rd and 5th days of colitis induction; P 0.01). The DAI was also significantly higher in the colitis group between days 2 and 6 of colitis induction when compared to the normal control and kefir-control groups. The DAI was statistically higher only on the 6th day in the kefircolitis group when compared to that in the normal control groups. Increased colon weight and decreased colon length were observed in colitis-induced rats. Mean colon length in the colitis group was significantly shorter than that of the kefir-control group. Kefir treatment significantly decreased histologic colitis scores(P 0.05). MPO activity in the colitis group was significantly higher than in the kefir-control group(P 0.05). Kefir treatment significantly reduced the DSS colitis-induced TNF-α increase(P 0.01). No statistically significant differences were observed among groups for IL-10 and MDA levels. Colon tissue i NOS levels in the colitis group were significantly higher than those in the control and kefir-colitis groups(P 0.05).CONCLUSION: Kefir reduces the clinical DAI and histologic colitis scores in a DSS-induced colitis model,possibly via reduction of MPO,TNF-α,and i NOS levels.