Objective To investigate the effect of GABAB receptor in spinal cord by detecting semi-quantitatively the mRNA for isoforms of GABAB receptor in a chronic neuropathic pain model. Methods Twenty-four male SD rats weighing 200-250g were anesthetized with intraperitoneal pentobarbital. Lamina of L5 vertebra and superior articular process of L6 were removed and posterior root of left L5 spinal nerve was exposed and tightly ligated with 7/0 atraumatic suture. The animals were randomly divided into 4 groups of 6 animals in each group: group A in which animals were observed for 7 days after sham operation; group B in which animals were observed for 7 days after ligation of posterior root of L5 spinal nerve; in group C animals were observed for 14 days after sham operation and in group D animals were observed for 14 days after ligation. Pain threshold was measured by heat stimulation of bilateral hindlimbs before and on the 1st , 3rd , 7th , 10th and 14th day after operation. Lumbar spinal cord was removed on the 7th or 14th day after operation and mRNA for isoforms of GABAB receptor in the spinal cord was detected semi-quantitatively by digoxin hybridization in situ.Results Pain threshold of left hindlimb (ligated side) was significantly lower than that of right hindlimb on the 7th, 10th and 14th day after operation as measured by heat stimulation. There were less mRNA for three isoforms of GABAB receptor in group B and D as compared with group A and C respectively. Conclusion The gene expression of three isoforms of GABAB receptor in the spinal cord has plasticity and is reduced during chronic neuropathic pain.

Objective:To explore the interaction between gossypol and human serum albumin ( HSA) . Methods:The interaction of gossypol and HSA under physiological conditions was studied by fluorescence spectroscopy, and the molecular docking software was used to simulate the interaction. Results:The binding constant of gossypol and HSA at 293K and 303K was 2. 390 6 × 105 L·mol-1 and 3. 576 8 × 103 L·mol-1 , respectively. There was one binding site on HSA for gossypol. Hydrogen bond and Van Der Waals inter-actions were involved in the binding process. The binding of gossypol and HAS was closer to tyrosine residue in HSA. The molecular simulation analysis verified the above results. Conclusion: The gossypol-induced fluorescence quenching of HSA belongs to a static quenching procedure.

This study examined the mechanism by which the gastric cancer cells lead to early peritoneal metastasis. HMrSV5 cells, a human peritoneal mesothelial cell line, were co-incubated with the supernatants of gastric cancer cells. Morphological changes of HMrSV5 cells were observed. The cell damage was quantitatively determined by MTT assay. The apoptosis of HMrSV5 cells was observed under transmission electron microscope. Acridine orange/ethidium bromide-stained condensed nuclei was detected by fluorescent microscopy and flow cytometry. The expressions of Bcl-2 and Bax was immunochemically evaluated. The results showed that conspicuous morphological changes of apoptosis were observed in HMrSV5 cells 24 h after treatment with the supernatants of gastric cancer cells. The supernatants could induce apoptosis of HMrSV5 cells in a time-dependent manner. The supernatants could up-regulate the expression of Bax and suppress that of Bcl-2 in HMrSV5 cells. These findings demonstrated that gastric cancer cells can induce the apoptosis of HPMCs through supernatants in the early peritoneal metastasis. The abnormal expressions of Bcl-2 and Bax may contribute to the apoptosis. Anti-apoptosis drugs promise to be adjuvant chemotherapeutic agents in the treatment of peritoneal metastasis of gastric cancer.
Apoptosis ; Cell Line ; Cell Line, Tumor ; Coculture Techniques ; Epithelial Cells/*cytology ; Epithelium ; Peritoneal Neoplasms/pathology ; Peritoneal Neoplasms/*secondary ; Stomach Neoplasms/*pathology

Objective To explore the effects and mechanisms of periostin overexpression on migration and invasion of nasopharyngeal carcinoma ( NPC) cell line.Methods The recombinant plasmids [ pCMV-neo ( +)-periostin ] and control plasmids [pCMV-neo (+)] were transfected into 6-10B cells using lipofectamine 2000TM reagent.The expression of periostin was detected with PCR and Western blotting .Transwell chamber invasion assay was employed to assay the migration and invasion of 6-10B cells before and after transfection .A gelatin zymogram was used to detect the activity of MMP-2 and MMP-9 in cultivated supernatant of 6-10B cells before and after transfection .The expression of integrin-αvβ5 was detected by immunohistochemistry ( IHC) in 6-10Bperiostin cells, 6-10Bvector cells and 6-10B cells as well as normal nasopharyngeal mucosa ( NNM) and NPC and at the same time periostin also was detected by immumohistochemistry in NNM and NPC, and densitometry analysis using image-pro plus 6.0 software, and the correlation between periostin and integrin-αvβ5 on NPC was assayed with statistics .Results Over expression of periostin promoted cell migration and invasion.The expression levels of integrin-αvβ5 in primary NPC and 6-10Bperiostin cells were significantly higher than those in NNM and 6-10Bvector, 6-10B cells.The expression in NPC of integrin-αvβ5 showed positively correlated with the expression of periostin (r=0.682, P<0.01).Conclusion Periostin plays an important role in regulation of cell migration and invasion probably by combining with integrin-αvβ5 to improve the activities of MMPs .

Alismatis Rhizoma(AR)is widely used in Chinese medicine,and its major bioactive components,tri-terpenes,reportedly possess various pharmacological activities.Therefore,it is very important to study the metabolism of triterpenes in vivo.However,the metabolism of AR triterpene extract has not been comprehensively elucidated due to its complex chemical components and metabolic pathways.In this study,an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method,which was based on the characteristic ions from an established database of known triterpenes,was used to analyze the major metabolites in rats following the oral administration of Alismatis Rhizoma extracts(ARE).As a result,a total of 233 constituents,with 85 prototype compounds and 148 metabo-lites,were identified for the first time.Hydrogenation,oxidation,sulfate and glucuronidation conjugation were the major metabolic pathways for triterpenes in AR.In addition,the mutual in vivo transformation of known ARE triterpenes was discovered and confirmed for the first time.Those results provide comprehensive insights into the metabolism of AR in vivo,which will be useful for future studies on its pharmacodynamics and pharmacokinetics.Moreover,this established strategy may be useful in meta-bolic studies of similar compounds.

Objective To investigate the potential protective effects of valproic acid (VPA) on gut barrier function after major burn injury in rats and its mechanism.Methods Forty male Sprague-Dawley (SD) rats were divided into sham + normal saline (NS),sham + VPA,scald + NS,and scald + VPA groups,with 10 rats in each group.Rat with 55％ total body surface area (TBSA) third-degree severe-bums model was reproduced by immersing into 80 ℃ water,and the rats in sham groups were given sham-bums by immersing into 37 ℃ water.The rats after severebums were immediately treated with 0.25 mL of 300 mg/kg VPA or NS by subcutaneous injection.Rats were sacrificed at 2 hours and 6 hours after injury,and abdominal aortic blood and ileal tissue were harvested.The levels of vascular endothelial growth factor (VEGF) were determined by enzyme-linked immunosorbent assay (ELISA).The intestinal permeability was evaluated by fluorescein isothiocyanate-dextran (FITC-dextran) determination.The histomorphological changes in gut barrier were evaluated by Chiu grading system.Levels of acetylated lysine at the ninth position of histone 3 protein (Ac-H3K9),hypoxia-inducible factor 1α (HIF-1α),zona occludens 1 (ZO-1) and myosin light chain kinase (MLCK) were determined by immunofluorescence staining and Western Blot.Results Compared with sham + NS group,rats in scald + NS group showed intestinal mucosal damage 2 hours after bum injury,as well as increased mucosal permeability,protein expression levels of HIF-1 α,VEGF,MLCK,and lowered levels of AC-H3K9 and ZO-1.These changes were much more prominent at 6 hours after injury.VPA treatment significantly attenuated the bum-induced intestinal damage.Compared with scald + NS group,the protective effects in scald + VPA group was not evident at 2 hours after injury;however,intestinal damage was much less severe at 6 hours after injury (Chiu score:2.03 ± 0.27 vs.3.12 ± 0.15),intestinal permeability was significantly decreased [FITC-dextran (μg/L):709 ± 76 vs.1138 ± 75],histone acetylation was enhanced [Ac-H3K9 (gray value):1.55 ± 0.12 vs.0.48±0.12],ZO-1 degradation was significantly inhibited (gray value:0.69 ± 0.12 vs.0.43 ± 0.16),the protein expression levels of VEGF and MLCK were significantly down-regulated [VEGF (ng/mg):51.7±3.7 vs.71.2±4.3,MLCK (gray value):1.98±0.20 vs.2.80±0.24],while the HIF-1 α protein expression levels were significantly reduced at both 2 hours and 6 hours after injury (gray value:2.50±0.39 vs.3.88±0.42 at 2 hours,1.83±0.42 vs.4.42±0.41 at 6 hours,all P ＜ 0.05).Conclusions Severe bum injury can induce histone deacetylation,ZO-1 degradation and intestinal barrier dysfunction.VPA can improve the levels of histone acetylation and ZO-1,and protect intestinal epithelial barrier function.These may probably be mediated through inhibiting HIF-1α and its downstream gene VEGF and MLCK.

OBJECTIVE:To establish the method for the simultaneous determination of content of 6 active components as neochlorogenic acid,mulberroside A,chlorogenic acid,astragalin,sanggenon C and morusin in Morus alba,and to provide reference for improving quality control standard of M. alba. METHODS:HPLC method was adopted. The determination was performed on Agilent 5 TC-C18with mobile phase consisted of acetonitrile-0.1% formic acid(gradient elution)at the flow rate of 1 mL/min. The detection wavelength of 280 nm. RESULTS:The mass concentration linear range of neochlorogenic acid, mulberroside A,chlorogenic acid,astragalin,sanggenon C and morusin were 0.001 06-0.042 4,0.001 67-0.066 8,0.007 95-0.318, 0.001 65-0.066 0,0.005 00-0.200 and 0.001 24-0.049 6 mg/mL,respectively(all r≥0.999 6);the limits of quantitation were 0.11, 0.14,0.81,0.17,0.45 and 0.12 μg/mL,respectively;the limits of detection were 0.04,0.05,0.41,0.07,0.18 and 0.04 μg/mL, respectively;RSDs of precision test were 0.26%,0.31%,0.24%,0.27%,0.36% and 0.44%(n=6),respectively;RSDs of stability test were 0.68%,0.54%,0.62%,0.53%,0.41% and 0.73%(n=6),respectively;average method recovery rates were 99.1%,98.8%,98.8%,98.4%,98.5% and 99.9%(RSDs were 0.5%-1.5%,n=9),respectively. CONCLUSIONS:The method is simple,accurate,and can be used for simultaneous determination of 6 active components in M.alba.

This study examined the mechanism by which the gastric cancer cells lead to early peritoneal metastasis.HMrSV5 cells,a human peritoneal mesothelial cell line,were co-incubated with the supernatants of gastric cancer cells.Morphological changes of HMrSV5 cells were observed.The cell damage was quantitatively determined by MTT assay.The apoptosis of HMrSV5 cells was observed under transmission electron microscope.Acridine orange/ethidium bromidestained condensed nuclei was detected by fluorescent microscopy and flow cytometry.The expressions of Bcl-2 and Bax was immunochemically evaluated.The results showed that conspicuous morphological changes of apoptosis were observed in HMrSV5 cells 24 h after treatment with the supematants of gastric cancer cells.The supernatants could induce apoptosis of HMrSV5 cells in a time-dependent manner.Th esupematants could up-regulate the expression of Bax and suppress that of Bcl-2 in HMrSV5 cells.These findings demonstrated that gastric cancer cells can induce the apoptosis of HPMCs through supernatants in the early peritoneal metastasis.The abnormal expressions of Bcl-2 and Bax may contribute to the apoptosis.Anti-apoptosis drugs promise to be adjuvant chemotherapeutic agents in the treatment of peritoneal metastasis of gastric cancer.

Objective:To correlate neoadjuvant radiotherapy and chemotherapy efficacy with changes in peripheral blood T lymphocytes in patients with advanced mid-to-low rectal cancer.Methods:A total of 106 patients with rectal cancer who received treatment in Jinhua Municipal Central Hospital from January 2019 to December 2020 were included in this study. Fasting venous blood was taken before neoadjuvant radiotherapy and chemotherapy and 7 days before surgery to measure the numbers of CD3 +, CD4 +, CD8 +, CD45RA + and CD45RO + cells using flow cytometry. The optimal cut-off point was determined using the receiver operating curve. The influential factors of tumor regression grade were analyzed using logistic regression analysis. Results:After neoadjuvant radiotherapy and chemotherapy, the numbers of CD3 +, CD4 +, CD8 + cells were (401.86 ± 138.65), (225.83 ± 87.17), and (155.84 ± 71.19) respectively, which were significantly decreased compared with before neoadjuvant radiotherapy and chemotherapy [(477.33 ± 141.74), (647.38 ± 203.19), (348.22 ± 113.75), t = 10.78, 11.17, 9.49, all P < 0.05]. There were no significant differences in the percentages of CD3 +, CD4 +, CD8 +, CD45RA + and CD45RO + cells between before and after treatment (all P > 0.05). The percentage of CD45RO + cells was significantly increased after neoadjuvant radiotherapy and chemotherapy. A higher percentage of CD45RO + cells led to a lower tumor regression grade ( P < 0.05). The receiver operating characteristic curve showed that the optimal cut-off point of the percentage of CD45RO + cells was 1.08. The area under the receiver operating characteristic curve was 0.774 ( P = 0.029), with a sensitivity of 82.5% and specificity of 69.6%. The logistic regression analysis revealed that the percentage of CD45RO + cells was significantly correlated with tumor regression grade ( P < 0.05). Conclusion:The percentage of CD45RO + cells in T lymphocyte subsets before and after neoadjuvant radiotherapy and chemotherapy is closely related to tumor regression grade. It can be used as an indicator to predict the sensitivity of neoadjuvant radiotherapy and chemotherapy. This study is of great innovation and science and provides a new idea for clinical practice.