Effect of electroacupunture on gastric mucosal intestinal trefoil factor gene expression of stress-induced gastric mucosal injury in rats

AIM： To investigate electroacupunture（EA） at the acupoints of Stomach Meridian of Foot-Yangming（SMFY）, Gallbladder Meridian of Foot-Yangming（SMFY） on gastric mucosal intestinal trefoil factor （ITF） gene expression detection in stress-induced rats with gastric mucosal lesion, and to explore the regulatory mechanism and significance of EA-related gastric mucosal protective effect. METHODS： Forty rats were randomly divided into 4 groups： Blank group, Model group, Model group＋EA at acupoints of SMFY group（＂SMFY group＂）, and Model group＋EA at acupoints of GMFY group（GMFY group）. All rats （except blank group） were made model by water immersion and restraint stress （WRS）. Then the gastric mucosa tissue in each rat was taken off alter assessment of gastric mucosal lesion index（GUI）, and the expression of ITF mRNA of the tissues was detected by reverse transcdption-polymerase chain reaction（RT-PCR） method. RESULTS： Compared with Model group（S4.3± 1.34）, the GUI value in SMFY group （31±2.211 decreased significantly（P〈0.01）, so did that in GMFY group （39.8± 1.62, P〈0.05）, meanwhile GUI value in SMFY group was significantly lower than in GMFY group（P〈0.01）. Compared with Model group （0.65±0.01）, EA had a tendency to improve the expression of gastric mucosal ITFmRNA gene： such tendency existed in GMFY group （0.66±0.01） but with no signficant difference（P〉 0.05）, in SMFY group（0.76±0.01） with an extremely obvious difference （P〈0.01）, furthermore the expression in SMFY group was significantly higher than in GMFY group （P〈 0.01）.CONCLUSION： The gastric mucosal protective effect by EA at the acupoints of SMFY and GMFY was related to the expression variance of ITF, indicating certain meridian specificity exists, It could be one proof for the TCM theory ＂Relative pardcularity between SMFY and stomach＂.

Pretreatment with intestinal trefoil factor alleviates stress-induced gastric mucosal damage via Akt signaling

BACKGROUND Stress-related gastric mucosal damage or ulcer remains an unsolved issue for critically ill patients.Stress ulcer prophylaxis has been part of routine intensive care,but uncertainty and controversy still exist.Co-secreted with mucins,intestinal trefoil factor(ITF)is reported to promote restitution and regeneration of intestinal mucosal epithelium,although the mechanism remains unknown.AIM To elucidate the protective effects of ITF on gastric mucosa and explore the possible mechanisms.METHODS We used a rat model of gastric mucosal damage induced by water immersion restraint stress and lipopolysaccharide-treated human gastric epithelial cell line to investigate the potential effects of ITF on damaged gastric mucosa both in vivo and in vitro.RESULTS ITF promoted the proliferation and migration and inhibited necrosis of gastric mucosal epithelia in vitro.It also preserved the integrity of gastric mucosa by upregulating expressions of occludin and zonula occludens-1.In the rat model,pretreatment with ITF ameliorated the gastric mucosal epithelial damage and facilitated mucosal repair.The protective effects of ITF were confirmed to be exerted via activation of Akt signaling,and the specific inhibitor of Akt signaling LY249002 reversed the protective effects.CONCLUSION ITF might be a promising candidate for prevention and treatment of stressinduced gastric mucosal damage,and further studies should be undertaken to verify its clinical feasibility.

Effects of Intestinal Trefoil Factor on Colonic Mucosa in Experimental Colitis of Rats

In order to investigate the protective effects of intestinal trefoil factor (ITF) on colonic mucosa in experimental colitis of rats, ITF was detected by RT-PCR and immunohistochemistry at different time points. Three days after colitis induction, rats were treated with either 0.9 % saline solution or rhITF. Pathological changes and the expression of iNOS mRNA, NO, MDA and SOD were measured respectively. It was found that ITF was mainly located in goblet cells, significantly higher in model group than in normal group (P<0.05). rhITF could increase the iNOS mRNA expression and NO contents, and there was statistically significant difference between rhITF group and model group (P<0.05). rhITF also caused an increase of MDA and a decrease of SOD, but there was no significant difference between two groups. These results indicated that ITF has apparent therapeutic effects in ulcerative colitis, which may be associated with iNOS and NO.

Production of human intestinal trefoil factor in Pichia pastoris

Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide was modified with a polybistidine tag sequence at the N-terminal, and then inserted into the P. pastoris expression vector pGAPZaA at the downstream of the s-mating factor signal. After gene sequencing, the recombinant pGAPZaA-hITF was transformed into the P. pastoris strain X-33 with lithium chloride, rhITF was induced to constitutively express in shake flask, and then analyzed with Tricine SDS-PAGEand Western blotting. The obtained rhITF was isolated from the cultured supernatants by ammonium sulfate precipitation, Ni-NTA affinity chromatography, and ultrafiltration. Results：The correctness and integrity of rhITF were identified by restriction digestion and gene sequencing, rhITF was successfully expressed to 50 mg/L as a secretive protein. After purification, the purity was above 95%. Tricine SDS-PAGE and Western-blot analysis showed that rhITF presented as a single band with a molecular weight of 10 kDa, a little larger than 7 879 Da as assayed by mass spectrometry analysis. Conclusion： hITF P. pastoris expression vector is successfully constructed and rhITF is expressed in P. pastoris at commercially relevant level. This research lays foundation for the further functional studying of hITF.

Synergistic Anti-inflammatory Effect of Radix Platycodon in Combination with Herbs for Cleaning-heat and Detoxification and Its Mechanism

Objective： To investigate the synergistic anti-inflammatory effect of Radix Platycodon in combination with herbs for cleaning-heat and detoxification and its mechanism for Fei （肺）-targeting. Methods： Forty Wistar rats were randomly divided into five groups （8 per group）： the sham-operated group, model group, Radix P/atycodon group, Flos Lonicera and Fructus Forsythia （LF） group, and Radix Platycodon, Flos Lonicera and Fructus Forsythia combination （PLF） group, using a random number table. A rat chronic obstructive pulmonary disease （COPD） model was established by passive smoking and intratracheal instillation of lipopolysaccharide （LPS）. The treatments started from the 15th day of passive smoking for a total duration of 14 days. At the end of the treatment, changes in the following measurements were determined： lung histopathology, inflammatory cytokines including tumor necrosis factor α （TNF- α）, transforming growth factor 13 （TGF- 13 ） and interleukin IL-1 13 （IL-1 13 ） in bronchoalveolar lavage fluid （BALF）, and mRNA expression of endogenous active substance intestinal trefoil factor 3 （TFF3） in the lung tissue. Results： Light microscopy showed that compared with the sham-operated group, rats in the COPD model group had disrupted alveolar structure, collapsed local alveoli, significantly widened or even fused alveolar septa, and massive infiltration of inflammatory cells in the alveolar wall and interstitium. In addition, significant bronchial epithelium hyperplasia, partially shed epithelia, and marked inflammatory cell infiltration in the bronchial wall and its surrounding tissues were noticed. Electron microscopy showed that rats in the model group had degeneration of alveolar type Ⅱ epithelial cell; reduction, breakage or even loss of cell surface microvilli; swollen mitochondria with disappearing cristae and vacuole-like structure; and, increased secondary lysosomes in alveolar macrophages. The TNF- α, TGF- β and IL-β levels and white blood cell （WBC） count in BALF were significantly increased （P〈0.01 or P〈0.05） and TFF3 mRNA expression in the lung tissue was significantly reduced （P〈0.01）. After treatment, the pathological morphology of lung injury was less severe in all three treatment groups. In addition, TGF- 13 and IL-1 13 and WBC count in BALF were decreased （P〈0.01 or P〈0.05）, and TFF3 mRNA expression in the lung tissue was significantly increased in the PLF group （P〈0.01）. Compared with the LF group, the IL-1 13 in BALF was significantly decreased （P〈0.05）, and TFF3 mRNA expression was significantly increased （P〈0.05） in the PLF group. Conclusions： Radix Platycodon synergizes with herbs for cleaning-heat and detoxification in reducing inflammatory injury in a rat model of COPD. The synergistic anti-inflammatory effect is reflected in the improvement in pathological changes and in the reduction of IL-1 β levels in BALF. The mechanism of such synergistic action may be related to its effect on maintaining the TFF3 mRNA expression and Fei-targeting function.