To investigate the biological role of snake venom cystatin(sv-cystatin) in tumor progression, the cDNA of sv-cystatin amplified by PCR from pUC18-cystatin plasmid was cloned into methanol-inducible expression vector pPICZ?A. The linearized recombinant plasmid pPICZ?A-cystatin was transfered into Pichia pastoris, strain GS115 by electrophoration. Transfermants with phenotype Mut+ selected were identified by PCR analysis and induced in 1.0% methanol. The reombinant sv-cystatin protein was examined by SDS-PAGE, Western blot analysis. The molecular mass of expression product was about 14 kDa and approximately 16 mg/L of recombinant sv-cystatin was produced from one of GS115-cystatin transformants. The chromatography purified protein could reduce the activity of papain. The ability of B16F1 cells treated with recombinant sv-cystatin to invade the reconstituted basement membrane decreased significantly (P

Objective To evaluate the clinical effect of computed tomography?guided visceral nerve plexus ethanol neurolysis through post?curs of diaphragm approach in the treatment of patients with pancreatic cancer pain using,and study the safety and life quality improvement of patients . Methods A total of 58 patients suffered from pancreatic cancer pain,who were treated in the department of pain medicine of the First Hospital of China Medical University from October 2013 to December 2014,were recruited for the study. The patients were divided into two groups according to the willing of the patients and their families,group A(32 cases)was treated with Visceral Nerve Plexus ethanol Neurolysis,while group B(26 cas?es)was treated with oral opioid drugs. The analgesic effect,changes in the amount of opioid drugs,changes in the PSQI scores and the improvement of quality of life were evaluated before treatment and 1 day(T1),15 days(T15),30 days(T30),60 days(T60)after treatment. Record the adverse reactions in the course of treatment. Results All the patients of group A successfully received visceral nerve plexus ethanol neurolysis,the VAS scores,Karnofsky scores,and PSQI scores of all the observed time points(after the operation)were statistically different compared to those before treatment and group B. Statistically difference was also observed in quality of life between two groups(P<0.05). The amount of opioid drugs of group B was statistically increased than that of group A(P<0.01).The most common side effects in Group A were postural hypotension(6 cases),diar?rhea(2 cases),and intercostal neuralgia,while nausea(20 cases),constipation(11 cases)and dizziness(8 cases)were seen in the Group B. Con?clusion Visceral nerve plexus ethanol neurolysis through post?curs of diaphragm approach by the guide of CT is effective and safe for the patients with pancreatic cancer pain,and the complications were totally acceptable.

Objective:To investigate the function of gasdermin D (GSDMD) in intestinal damage of mice with severe acute pancreatitis (SAP).Methods:The healthy C57BL/6 mice were divided into four groups randomly, including normal saline (NS) group, small interfering RNA (siRNA)-NS group, SAP model group and siRNA-SAP group, with 6 mice in each group. The SAP mouse model was reproduced by intraperitoneal injection of caerulein 50 μg/kg combined with lipopolysaccharide (LPS) 10 mg/kg; the NS group was given the same amount of NS; in the siRNA-SAP group and siRNA-NS group, siRNA 50 mg/kg was injected through the tail vein three times before modeling or injection of NS. The blood of mice eyeball in each group was taken 12 hours after modeling, and serum interleukins (IL-1β, IL-18) levels were detected by enzyme linked immunosorbent assay (ELISA). The mice were sacrificed to observe the general changes in abdominal cavity, the pancreas and ileum tissues were taken to observe the pathological changes under a light microscope. The expression of long-chain non-coding RNA uc.173 (lnc uc.173) was detected by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical method was used to detect the expression of tight junction proteins zonula occluden-1 (ZO-1) and Occludin in intestinal mucosal epithelial cells. Western blotting was used to detect the GSDMD protein expression level in the intestinal tissue.Results:The serum levels of IL-1β and IL-18 in the SAP model group were significantly higher than those in the NS group and the siRNA-NS group [IL-1β (ng/L): 146.66±1.40 vs. 44.48±5.76, 81.49±10.75, IL-18 (ng/L): 950.47±177.09 vs. 115.43±16.40, 84.84±21.90, all P < 0.05]; and the levels of IL-1β and IL-18 in the siRNA-SAP group were significantly lower than those in the SAP model group [IL-1β (ng/L): 116.26±15.54 vs. 146.66±1.40, IL-18 (ng/L): 689.96±126.08 vs. 950.47±177.09, both P < 0.05]. General observation showed that there were no obvious abnormalities in the abdominal cavity of the mice in the NS and siRNA-NS groups; the mice in the SAP model group and the siRNA-SAP group had different degrees of edema and congestion in the intestine; compared with the SAP model group, the abnormalities in the siRNA-SAP group was significantly reduced. Under light microscope, there were no obvious changes in the pancreas and intestinal mucosa in the NS group and the siRNA-NS group; the pancreatic tissue of the SAP model group and the siRNA-SAP group had different degrees of edema, inflammatory cell infiltration, and lobular structure damage, and the intestinal mucosa was damaged to a certain degree, and the villi were broken to varying degrees, but the damage in the siRNA-SAP group was lighter. The results of RT-PCR showed that the expression of lnc uc.173 in the intestinal tissues of the model SAP group was significantly lower than that of the NS group and the siRNA-NS group (2 -ΔΔCt: 0.26±0.12 vs. 1.01±0.37, 0.67±0.32, both P < 0.05), while the expression of lnc uc.173 in the siRNA-SAP group was significantly higher than that in the SAP model group (2 -ΔΔCt: 0.60±0.39 vs. 0.26±0.12, P < 0.05). Immunohistochemistry showed that ZO-1 and Occludin proteins in the NS group were distributed along the epithelial cells of the intestinal mucosa, showing a strong expression; ZO-1 and Occludin expressions were significantly reduced in the SAP model group and siRNA-SAP group, but the expressions in the siRNA-SAP group was higher than that in the SAP model group. Western blotting showed that the expression level of GSDMD protein in the intestinal tissues of the SAP model group was significantly higher than that of the NS group and the siRNA-NS group [GSDMD protein (GSDMD-N/β-actin): 1.99±0.46 vs. 1, 1.00±0.78, both P < 0.05]. Compared with the SAP model group, the expression of GSDMD protein in the siRNA-SAP group was significantly decreased [GSDMD protein (GSDMD-N/β-actin): 1.42±0.42 vs. 1.99±0.46, P < 0.05]. Conclusions:The systemic inflammatory response and intestinal mucosal barrier damage of SAP mice may be related to the increase of GSDMD expression in intestinal tissues. GSDMD mediates cell pyrolysis to promote the release of inflammatory factors, cause intestinal injury, and down-regulate the expression of intestinal epithelial cell tight junction proteins such as ZO-1 and Occludin, resulting in intestinal mucosal damage.

OBJECTIVETo observe the regulatory effects of psoralen, oleanolic acid, and stilbene glucoside, three active components of psoralea fruit, glossy privet fruit and tuber fleeceflower root respectively, on Aβ25-35induced self-renewal and neuron-like differentiation of neural stem cells (NSCs).

METHODSEmbryonic NSCs werein vitro isolated and cultured from Kunming mice of 14-day pregnancy, and randomly divided into the control group, the Aβ25-35 group, the Aβ25-35 +psoralen group, the Aβ25-35 +oleanolic acid group, and the Aβ25-35 + stilbene glucoside group. The intervention concentration of Aβ25-35 was 25 µmol/L, and the intervention concentration of three active components of Chinese medicine was 10(-7)mol/L. The effect of three active components of Chinese medicine on the proliferation of NSCs was observed by counting method. The protein expression of Tubulin was observed by Western blot and immunofluorescence. The ratio of Tubulin+/DAPI was caculated. Results Compared with the control group, the sperical morphology of NSCs was destroyed in the Aβ25-35 group, the counting of NSCs, the expression of Tubulin protein, and the ratio of Tubulin /DAPI all decreased (P <0.01, P <0.05). Compared with the Aβ25-35 group, the counting of NSCs, the expression of Tubulin protein, and the ratio of Tubulin + /DAPI all increased in the three Chinese medicine treated groups (P <0. 01, P <0. 05).

CONCLUSIONS25 µmol/L Aβ25-35 could inhibit self-renewal and neuron-like differentiating of NSCs. But psoralen, oleanolic acid, and stilbene glucoside could promote self-renewal of NSCs and neuron-like differentiation.

Amyloid beta-Peptides ; physiology ; Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Embryo, Mammalian ; Female ; Mice ; Neural Stem Cells ; Neurogenesis ; drug effects ; Neurons ; cytology ; Peptide Fragments ; physiology ; Pregnancy

Sulfur fumigation, which is traditional method for preservation, pest control, insecticide and sterilization, has long been widely used in processing and storage and played a positive role of traditional Chinese medicine (TCM). As some businesses sided pursuit of profit, abused and repeated use of sulfur fumigation, have resulted in a large number of harmful residues, such as sulf dioxide (SO2) and harmful heavy metals, which brings a significant impact and danger on human health. This article summarizes the sulfur species and the sulfur fumigation methods and analyzes the harmful substances in TCM after sulfur fumigation, to provide a reference of the choice of species for the sulfur, the optimization of sulfur fumigation process and the standardized processing of TCM after sulfur fumigation.
Animals ; Drug Contamination ; Fumigation ; methods ; Humans ; Medicine, Chinese Traditional ; methods ; Safety ; Sulfur ; chemistry ; Technology, Pharmaceutical ; methods

Objective To introduce an improved method for collecting the blood of rat by cardiac puncture .Methods The study se-lected 90 Wistar rats,half male and half female ,and then the rats were anesthetized by intraperitoneally injecting 10％chloral hydrate solution according to 0.3 mL/kg body weight.Then using three-line positioning method to determine the puncture site ,the blood collection needle and blood collection tube were used to collecting the blood ,Finally,the blood volume of each rat was recorded .Results The success rate was cal-culated according to the single blood collection of 5 mL or more,and our 90 rats were collected 90 times,the success rate was about 82.2％. After centrifugation,the total serum was about 260 mL,the average serum separation of each rat was about 2.8 mL.Conclusion The im-proved positioning method of heart blood collection is intuitive and easy to learn ,and the total volume and quality of blood is high ,besides ,the new method has a small surgical trauma and a high efficiency ,which is worth to extend .

OBJECTIVETo investigate the effect of siRNA silencing the role of C-Jun N-terminal Kinase (JNK) gene in excessive endoplasmic reticulum stress on lung ischemia/reperfusion injury.

METHODSMouse model of pulmonary ischemia reperfusion injury (PIRI) in situ was established with unilateral lung in vivo. Seventy experimental mice were randomly allocated into seven groups (n = 10): Sham group (Sham group), ischemia reperfusion group (I/R), PBS+ Lipofectamine2000TM transfection reagent group (I/R + PBS+ Lipo group), negative control group (I/R+ SCR group), JNK-siRNA group (I/R + siRNA(JNK1), siRNA(JNK2), siRNA(JNK3)). Mice were euthanized after experimental time out, and left lung tissue was extracted. Wet/dry lung weight ratio (W/D) and total lung water content (TLW) were tested. Light microscope, alveolar damage quantitative evaluation index (IQA) and electron microscope were observed. The expression levels of JNK and glucose regulatex protein(GRP78) were detected by RT-PCR and Western blot. Apoptosis of lung tissue was determined by TUNEL.

RESULTSCompared with Sham group, all indicators above of I/R + PBS + Lipo group and I/R + SCR group were significantly increased (P < 0.01), and compared with I/R group, those indicators of the three groups all had no notable difference; those indicators were not statistically different between I/R + PBS + Lipo group and I/R + SCR group, and compared to the three groups, the above indicators in JNK-siRNA group were lower (P < 0.05, P < 0.01) except that the expression levels of GRP78 was not statistically different.

CONCLUSIONI/R induces excessive ERS in lung tissue, in which JNK pathway participates in apoptosis, leading to lung tissue injury.

Animals ; Apoptosis ; Endoplasmic Reticulum Stress ; Heat-Shock Proteins ; metabolism ; JNK Mitogen-Activated Protein Kinases ; genetics ; Lung ; physiopathology ; Lung Injury ; genetics ; MAP Kinase Signaling System ; Mice ; RNA, Small Interfering ; Reperfusion Injury ; genetics

AIM:To identify the expression of fermitin family homolog 2 (FERMT2) in hepatocellular carci-noma ( HCC) tissues and the effect of FERMT 2 on the cell growth and related protein expression .METHODS:Real-time PCR and immunohistochemistry were used to detect FERMT 2 expression in the HCC tissues .The technique of CRISPR/Cas9 was applied to construct stable FERMT2 knockout MHCC97H cell line.WST-1 assay and flow cytometry were used to measure the cell viability , cell-cycle distribution and cell apoptosis .Western blot was used to determine the expression of related proteins in the MHCC97H cells.RESULTS:In HCC tissues, the expression level of FERMT2 was higher than that in adjacent liver tissues (P<0.05).High expression of FERMT2 was significantly correlated with postoperative recurrence of tumor.Knockout of FERMT2 gene evidently inhibited MHCC97H cell viability and accelerated cell apoptosis .Mean-while, the expression levels of proliferating cell nuclear antigen , cyclins, cyclin-dependent kinases 2 and anti-apoptotic fac-tors were significantly downregulated in MHCC97H cells with FERMT2 knockout (P<0.05).CONCLUSION:FERMT2 may function as a promoter of hepatocarcinogenesis and progression via regulating the cell viability , cell-cycle distribution and cell apoptosis , which is related with the expression of cell cycle regulators and anti-apoptotic factors .

Objective To evaluate the use of gated SPECT in patients with hypertrophic obstructive cardiomyopathy (HOCM) and the effects of percutaneous transluminal septal myocardial ablation (PTSMA) on myocardial perfusion.Methods 99 Tcm-methoxyisobutylisonitrile (MIBI) and 18F-fluorodeoxyglucose (FDG) images were performed in 31 HOCM patients before PFSMA and in 15 patients 3-7 d after PTSMA.The images in different left ventricular segments were analysed by using scores.Results In 99Tcm-MIBI images, uptake decreased at the septal regions in 12 HOCM patients (80.0%, 12/15) after PTSMA, 18F-FDG images also showed decreased uptake at the septal regions in 5 HOCM patients (33.3%, 5/15) after PTSMA.Conclusion 99Tcm-MIBI images might be an important method to evaluate PTSMA results, and 18 F-FDG images showed important value as reference.

OBJECTIVEInflammation serves as the initial pathologic step of cardiovascular diseases including atherosclerosis. Resveratrol possesses many pharmacological properties including antioxidant, cardioprotective and anti-cancer effects. In this study, we investigate the anti-inflammatory effect and mechanisms of resveratrol in an atherosclerotic rabbit model.

METHODSRabbit were assigned to six groups (n = 10 each): control, high fat diet group, resveratrol low, medium and high dose groups, resveratrol pretreatment group. The serum tumor necrosis factor-α (TNF- α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were analyzed by Enzyme-linked immuno sorbent assay(ELISA). Phosphorylation levels of mitogen-activated protein kinases (MAPKs) cascades and NF-κB were determined by Western blot.

RESULTSCompared with the control group, the expression of serum inflammatory factors IL-1β, IL-6, TNF-α were increased in high-fat group (all P < 0.05). Compared with high-fat group, the expressions of IL-6, IL-1β, TNF-α were significantly reduced in resveratrol low, medium, high dose groups and resveratrol pretreatment group (all P < 0.01), and this effect is dose-dependent. In addition, the NF-κB, p38MAPK, JNK, ERK1/2 protein phosphorylation in high-fat group were significantly upregulated compared with control group (P < 0.05), which (except ERK1/2 phosphorylation level) were significantly downregulated in resveratrol treatment group and resveratrol pretreatment group.

CONCLUSIONThis study indicates that resveratrol reduces serum inflammatory cytokines in this atherosclerotic rabbit model via down-regulation phosphorylation of NF-κB, and MAPKs signaling, which might serve as the anti-inflammatory molecular basis of resveratrol.

Animals ; Atherosclerosis ; drug therapy ; metabolism ; Disease Models, Animal ; Interleukin-1beta ; blood ; Interleukin-6 ; blood ; Male ; Mitogen-Activated Protein Kinases ; metabolism ; NF-kappa B ; metabolism ; Phosphorylation ; Rabbits ; Signal Transduction ; drug effects ; Stilbenes ; pharmacology ; therapeutic use ; Tumor Necrosis Factor-alpha ; blood