Objective To investigate the protective effects of elctroacupuncture(EA)at Zusanli(ST36) points on intestinal villas damage and mucosal permeability induced by small intestine pro-inflammatory factors in rats with intestinal ischemia/reperfusion(I/R). Methods 30 Sprague-Dawley(SD)rats were randomly divided into three groups(each,n=10):intestinal I/R group(model group),intestinal I/R+EA ST36 group(EA group)and intestinal I/R+sham EA group(SEA group). Rats were subjected to superior mesenteric artery(SMA)clamping at its root part to occlude the vessel for 30 minutes,followed by reperfusion for 60 minutes to form intestinal I/R models. Rats in EA group received EA at the bilateral ST36 points(2-3 mA,2-100 Hz)for 30 minutes immediately after ischemia,those in SEA group received EA at bilateral sham points(the point was located at 0.5 cm away from ST36 point in its lateral side)with the same frequency and intensity of stimulation as EA group for 30 minutes,and those in model group received no treatment. Animals were sacrificed 60 minutes after reperfusion and segments of distal part of ileum were harvested,then the levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in intestinal tissue were measured. Histopathologic changes were viewed and graded via light microscopy. A solution of fluorescein isothiocyanate(FITC)-dextran was injected into the lumen of the segment of intestine 30 minutes after reperfusion,systemic blood was drawn via abdominal aorta puncture at 60 minutes after reperfusion,and then the level of FITC-dextran in blood was measured to determine the changes in intestinal permeability. Results Compared to the model group and SEA group,EA ST36 significantly attenuated intestine TNF-α(pg/mg:3.01±0.50 vs. 8.65±1.02,8.42±1.41,both P<0.05)and IL-6 levels(pg/mg:2.51±0.15 vs. 6.34±0.86,6.13±1.12,both P<0.05),successfully maintained low gut injury scores(1.50±0.33 vs. 3.18±0.39,3.04±0.37,both P<0.05), and significantly reduced permeability of the distal ileum and the content of FITC-dextran(μg/L:282.42±73.92 vs. 856.22±229.47,844.22±239.47,both P<0.05). However,there were no significant differences in all above variables between SEA and model group(all P>0.05). Sections of distal ileum from animals in the model group and SEA group showed no obvious difference histologically,and the pathological manifestations were villous tip necrosis, blunt-shaped and collapse. Compared to the model group and SEA group,the intestinal villous injury in animals of EA group was much milder. Conclusion In rats with intestinal I/R injury,EA ST36 points has protective effect on the gut that is possibly due to the fact it may obviously lower the levels of the pro-inflammatory factors of small intestinal tissue,alleviate mucosal insult of gut and reduce the mucosal permeability.

Acute Disease ; Animals ; Behavior, Animal ; drug effects ; Female ; Male ; Mice ; Mice, Inbred ICR ; Nitriles ; poisoning ; Rabbits

OBJECTIVEThe present study is concerning qualitative and quantitative detection of Poria cocos quality based on FT-near infrared (FT-NIR) spectroscopy combined with chemometrics.

METHODThe Poria cocos polysaccharides contents were determined by UV. Transmission mode was used in the collection of NIR spectral samples. The pretreatment method was first derivation and vector normalization. Then principal component analysis (PCA) was used to build classification model and partial least square (PLS) to build the calibration model.

RESULTThe results showed that conventional criteria such as the R, root mean square error of calibration (RMSEC), and the root mean square error of prediction (RMSEP) are 0.944 0, 0.072 1 and 0.076 2, respectively. The misclassified sample is 0 using the qualitative model built by PCA.

CONCLUSIONThe prediction models based on NIR have a better performance with high precision, good stability and adaptability and can be used to predict the polysaccharose content of Poria cocos rapidly, which can provide a fast approach to discriminate the different parts of Poria cocos.

Fungal Polysaccharides ; analysis ; Least-Squares Analysis ; Poria ; chemistry ; Principal Component Analysis ; Spectroscopy, Near-Infrared ; methods

Objective To establish a method of labeling human mesenchymal stem cells (MSCs) with PKH26 in vitro.Methods MSCs were cultured and labeled with PKH26 according to the manufacturer's instruction.The growth,fluorescence intensity and serial subcuhivation of labeled MSCs were analyzed with the confocal laser microscope and the flow cytometry.The biological characteristics of labeled MSCs were investigated by RT-PCR.Results The labeled MSCs appeared red fluorescence and the labeling rate was 100 percent.During serial subcuhivation of labeled MSC from passage 1 to passage 7,the fluorescence intensity and the labeling rate of MSCs were gradually decreased.The biological features such as morphology,growth,expression level of nucleostemin and GAPDH gene and capability of differentiation into osteoblast in vitro were not affected by labeling.Conclusion Labeling the human MSCs with PKH26 is an effective and practical method,which can be used as an important tool in the study on the homing, plasticity and transplantation of MSCs.

OBJECTIVETo screen the specific antibody binding peptides of tuberculosis from phage-displayed random phage display (Ph.D.)-7 peptide libraries with purified IgG from tuberculosis serum, and to provide the basis for the development of serological detection reagent of tuberculosis.

METHODSPurified IgG of tuberculosis serum was used as solid ligand to screen the binding peptide from the Ph.D.-7 peptide library, according to the biopanning process of absorption, elution and amplification. Purified IgG of health people serum was used as the molecule of counter selection during the second and third selection. Phages were enriched after 3 rounds of screening, then 20 single phages separately eluted by IgG of tuberculosis and health people were randomly selected on each direction of the determination plates and amplified. The single chains DNA were extracted as template for sequencing. The combination abilities of selected clones to IgG of tuberculosis and health people were tested by indirect enzyme linked immunosorbent assay (ELISA), and the positive clone was identified. Serum samples, from 47 patients with tuberculosis and 37 healthy people vaccinated with BCG, were verified by positive phage clones using phage-ELISA. The verifying results of 24 serum samples used for panning were separately analyzed statistically.

RESULTSAfter 3 rounds of panning, remarkable enrichment of phages that could specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 12 sequences were obtained. 12 phage clones with different sequences were amplified and detected with indirect ELISA and single phage H12 showed higher affinity with IgG of tuberculosis (S/N ≥ 2.1) and was identified as the positive clone. It was found that, in indirect ELISA with single Phage H12, the A(450) value of tuberculosis patients (0.105 ± 0.010) was significantly higher than that of healthy individuals (0.070 ± 0.005), and the t value was 2.912 (P < 0.0001). The A(450) value of 12 serum samples of tuberculosis patients and 12 samples of health individuals used for panning were 0.144 ± 0.016 and 0.052 ± 0.004, and the t value was 5.69 (P < 0.0001).

CONCLUSIONBy using phage-displayed random peptide libraries, we obtained the specific antibody binding peptides of tuberculosis, which showed specific binding activity with IgG of tuberculosis. It can provide a basis for the establishment of a new serological detection method of tuberculosis with peptide.

Adult ; Case-Control Studies ; Female ; Humans ; Immunoglobulin G ; blood ; Male ; Middle Aged ; Molecular Sequence Data ; Mycobacterium tuberculosis ; immunology ; Peptide Library ; Peptides ; immunology ; Tuberculosis ; diagnosis ; immunology ; microbiology ; Young Adult

A method was established for the simultaneous analysis of 25 trace elements and heavy metals in polysccharides from Liuwei Dihuang prescription, including Li, Be, B, Ti, Mg, Al, V, Cr, Mn, Co, Fe, Ni, Cu, Zn, Ga, As, Sr, Cd, Sn, Sb, Ba, Hg, Tl, Pb, Bi. The different rate of elemental extraction in Al, Fe, Mg, B, Ti, Mn, Zn, Sr, Ba was made in water and different concentration of alcohol. The samples, digested via microwave, calibrated by internal standard elements such as Ge and In, with bush branches and leaves as the controlled reference standard, were inlet into ICP-MS to analyze the contents of the 24 trace elements and heavy metals. The detection limits of the 24 elements were in the range of 0.007-2.225 µg · L(-1), while the RSD was below ≤ 4. 0%, with their recovery ranging from 84. 1% to 116%. Big different of the elemental extraction rates could be found by using different ethanol solutions. The method is simple, rapid and accurate, and can be used for the quality control of trace elements and heavy metals in Liuwei Dihuang polysccharides. With the aid of the obtained result, we may increase the extraction of necessary element while making an attempt at multi-element speciation in polysccharides from Liuwei Dihuang.
Chemical Fractionation ; Drugs, Chinese Herbal ; chemistry ; Mass Spectrometry ; methods ; Polysaccharides ; chemistry ; isolation & purification ; Trace Elements ; chemistry ; isolation & purification

OBJECTIVETo investigate if the interaction between TGF-beta1/Smad pathway and ERK pathway in vascular smooth muscle cells exists.

METHODSThe rat arota was removed. The primary VSMC were isolated and cultured in vitro, then the VSMC were divided into four groups: (1) control group, (2) (TGF-beta1 group, (3) ERK blocking agent group, (4) TGF-beta1 + ERK blocking agent group. The expression of Smad2/3, ERK1/2 proteins, the content of phosphorylated ERK1/2 and Smad2/3 proteins were detected by Western blot, and the expression of Smad2/3 mRNA was detected by reverse transcription-polymerase chain reaction(RT-PCR) .

RESULTS(1) In contrast to control group, the content of phosphorylated Smad2/3 and phosphorylated ERK1/2 proteins in TGF-beta1 group was increased (P < 0.05), that in ERK blocking agent group was decreased (P < 0.05). There was no difference between control group and TGF-beta1 + ERK blocking agent group. Compared with TGF-beta1 group, the contents of phosphorylated Smad2/3 and phosphorylated ERK1/2 proteins in TGF-beta1 + ERK blocking agent group was decreased (P < 0.05). There was no difference in the expression of Smad2/3 and ERK1/2 proteins among different groups. (2) There were no differences in expression of Smad2 and Smad3 mRNA among different groups.

CONCLUSION(1) TGF-beta1 can induce Smad2/3 proteins to be phosphorylated dependent on the activated ERK pathway. (2) ERK pathway does not effect the expression of Smad2/3 at the level of protein and mRNA.

Animals ; Aorta ; cytology ; Cells, Cultured ; Female ; MAP Kinase Signaling System ; physiology ; Male ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Muscle, Smooth, Vascular ; cytology ; physiology ; Myocytes, Smooth Muscle ; physiology ; Phosphorylation ; Rats ; Rats, Wistar ; Signal Transduction ; Smad Proteins ; metabolism ; physiology ; Transforming Growth Factor beta1 ; physiology

We investigated the effects of transient receptor potential M8 (TRPM8) channel on the proliferation and motility of androgen-independent prostate cancer PC-3 cells. After being permanently transfected with an empty vector and cDNA encoding the TRPM8 protein, cells were analysed for cell cycle distribution and motility using flow cytometry and scratch assay. Immunocytochemistry and Ca2+ imaging analysis revealed the overexpression of functional TRPM8 channel on both endoplasmic reticulum and plasma membrane of PC-3-TRPM8 cells. Cell cycle distribution and scratch assay analysis revealed that TRPM8 induced cell cycle arrest at the G0/G1 stage (P < 0.05) and facilitated the cell apoptosis induced by starvation (P < 0.05). Furthermore, TRPM8 inhibited the migration of PC-3-TRPM8 cells (P < 0.01) through the inactivation of focal-adhesion kinase. It appears that TRPM8 was not essential for the survival of PC-3 cells; however, the overexpression of TRPM8 had negative effects on the proliferation and migration of PC-3 cells. Thus, TRPM8 and its agonists may serve as important targets for the treatment of prostate cancer.
Adenocarcinoma ; genetics ; metabolism ; pathology ; Apoptosis ; Calcium ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; physiology ; Cell Proliferation ; Cell Transformation, Neoplastic ; Cytosol ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Epithelial Cells ; metabolism ; pathology ; Focal Adhesion Protein-Tyrosine Kinases ; antagonists & inhibitors ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Starvation ; pathology ; TRPM Cation Channels ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transfection

Objective:To investigate the effects of moxibustion on the serum metabolism in healthy human body based on the 1H nuclear magnetic resonance (1H NMR) metabolomics technology, and to find the differences in metabolites, as well as to elucidate the effects of moxibustion on healthy human body from the viewpoint of global metabolism. Methods:Sixty subjects of healthy young men from the enrolled students were randomly divided into a moxibustion group and a control group using random number table, with 30 cases in each group. Subjects in the moxibustion group accepted mild moxibustion on the right Zusanli (ST 36), once a day, 15 min for each time, and continuous treatment for 10 d; those in the control group did not receive any intervention. There were 28 cases in the moxibustion group and 23 cases in the control group after interventions. On the 1st day, 5th day and 10th day of the intervention, serum samples were collected from subjects of the two groups, and metabolic spectra were obtained by the1H NMR technology. Results: Before and after the intervention, serum1H NMR of the moxibustion group was significantly different, while the difference was insignificant in the control group. Metabolite changes in the moxibustion group were mainly in low density lipoprotein (LDL)/very low density lipoprotein (VLDL), valine, isoleucine, leucine, lactic acid, glutamine, citric acid, polyunsaturated fatty acids, creatine, glycine, glycerol, glucose, tyrosine, histidine, formic acid, alanine, lysine, acetic acid, and glutamic acid. Conclusion:Moxibustion can cause changes of serum metabolic patterns in healthy human by influencing the concentrations of branched-chain amino acids, polyunsaturated fatty acids, and other metabolites to strengthen body's metabolisms of amino acids and fatty acid.

OBJECTIVETo investigate the expression of inflammatory factors in lung tissue of acute paraquat (PQ) poisoned rats.

METHODSFifty male SD rats were randomized divided into two groups: the normal control group (NC group, n = 10) and the PQ group (n = 40). The 1 ml saline was administered once in normal control group. The PQ group was administered with 20 mg/kg 1% PQ by intraperitoneal injection to establish the model of PQ induced lung injury. At six hours, at the first, the third and the seventh day the PQ group were sacrificed, while at the first day the normal control group was sacrificed. The level of tumor necrosis factor alpha (TNF-alpha) mRNA, interleukin 10 (IL-10) mRNA, high mobility group box 1 (HMGB-1) mRNA in lung of rats were detected. Meanwhile, pathological changes of the lung were examined under optical microscope.

RESULTSCompared with that in normal control group, TNF-alpha mRNA expression in lung tissue of PQ group reached the peak at the six hour and decreased slowly at the first day [(0.740 +/- 0.100) and (0.584 +/- 0.049) respectively]. At the six hour and the first day in PQ group it was significantly higher than that in normal control group (P < 0.05 or P < 0.01). IL-10 mRNA expression in lung tissue of PQ group was elevated at the six hour, reached the peak at the first day, at the third day [(0.551 +/- 0.016) and (0.524 +/- 0.010) respectively] and the seventh day also higher than that in normal control group. At the first and the seventh day in the PQ group it was significantly higher than that in normal control group (P < 0.01). Meanwhile, HMGB-1 mRNA expression in lung tissue of PQ group was also elevated at the six hour, reached the peak at the first day, at the third [(0.695 +/- 0.060), (0.871 +/- 0.154) and (0.819 +/- 0.188) respectively] and the seventh day also higher than that in normal control group. At six hour, the first and the third day in the PQ group it was significantly higher than that in normal control group (P < 0.01). The histological changes such as alveolar edema, hemorrhage and inflammatory cell infiltration in the PQ group were more than those in the normal control group.

CONCLUSIONIn rats after PQ intoxication the levels of the inflammatory factors TNF-alpha, IL-10 and HMGB-1 are higher than normal rats, and inflammatory could play an important role in lung injury of poisoned rats.

Acute Disease ; Animals ; Disease Models, Animal ; HMGB1 Protein ; genetics ; metabolism ; Interleukin-10 ; genetics ; metabolism ; Lung ; drug effects ; metabolism ; pathology ; Male ; Paraquat ; poisoning ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; genetics ; metabolism