Objective:The aim of this study was to observe the role of NO in the action of tetramethylpyrazine (TMP) on arterioles vasomotion induced by noradrenaline (NA), in order to make further understand of vasodilation mechanism of TMP.Methods: Albino rabbits weighting 2.0 ～ 2.5 kg were anesthetized. They were ventilated with room air via tracheotomy tube. The arterial pressure was monitored with a pressure transducer attached to a polyethylene canula in the right common carotid artery by four channel polygraph, the intestinal loop was mounted on the stage of an inverted microscope and bathed in balanced Kreb Ringer solution maintained at 37.5 ?C . The inside diameter of arterioles were measured manully on a TV monitor using a TV camera mounted on the microscope. Observe the changes of BP、 microcirculation and filtration of true capillary after injection of TMP 8mg/kg、 N G monomethyl L arginine( L NMMA) 10mg via a cannula in the left carotid artery after topical application of NA(0.8?g) conducted in the measured field. Results:TMP can inhibit spontaneous vasomotion in vasodilation; L NMMA can inhibit it in vasoconstriction stage. L NMMA have no effect on the action of TMP. Conclusions: Endogenous NO has no role in TMP's vasodilation.

Objective To investigate the change in GABA receptor-activated current in dorsal root ganglion (DRG) neurons in rats with neuropathic pain. Methods Twenty adult SD rats of both sexes weighing 100-150 g were randomly divided into 2 gorups: sham operation group (group S, n = 5) and neuropathic pain group (group NP, n= 15). Neuropathic pain was induced by ligation of right L5 spinal nerve. The animals were sacrificed at 5 days after operation. The L5 DRG( neurons in group NP and L3-5 DRG neurons in group S were immediately isolated. Whole-cellpatch- clamp technique was used. The extracellular solution contained GABA 100μmol/L.The frequency and amplitude of the GABA-activated current in DRG neurons and the changes in action potential (threshold potential, rheobase and overshoot) and resting potential before and after GABA administration were recorded. Results GABA 100μmol/L induced rapid inactivation of inward current in most neurons. Compared with the baseline before application of GABA, in group S GABA induced depolarization,increased resting potential and decreased amplitude and rheobase of action potential in large and medium DRG neurons, while in group NP GABA increased resting potential but induced no significant change in threshold potential and rheobase and overshoot of action potential. The frequency and amplitude of GABA-activated current and the degree of change in resting potential and rheobase and overshoot of action potential were significantly lower in group NP than in group S.Spontaneous discharge occurred in small DRG neurons in both groups. No GABA-activated current was observed in all DRG neurons with spontaneous discharge. Conclusions Neuropathic pain is induced by decreasing GABA-mediated inhibition signals in large and medium DRG neurons leading to increased excitability of neurons.

AIM AND METHODS: To explore the effects calcitonin gene-related peptide (CGRP) and endothelin-1(ET-1) on the mechanisms of hypoxic pulmonary hypertension (HPH),the contents of CGRP and ET-1 in plasma of pulmonary artery and thoracic aorta and in extractives of lung and ventricular tissues of the chronic hypoxic rats were determined by radioimmunoassay. The changes of their hemodynamic indices and right heart hypertrophy index were monitored simultaneously. RESULTS: The level of pulmonary artery plasma CGRP was significantly higher than that of thoracic aorta plasma,but just the reverse was ET-1 or the ratio of ET-1 and CGRP in control rats( P

OBJECTIVETo study the inhibitory activity of Aeschynanthus maculatus on alpha-glucosidase.

METHODThe inhibilitory model of in vitro alpha-glucosidase was established. Active extracts of A. maculatus were isolated and identified bymultiple chromatographic methods, and their molecular structures were identifiied by spectral techniques.

RESULTSeven coumpounts were isolated from A. maculatus and isolated as lupeol(1), stigmasterol(2), ursolic acid(3), stigmast-5,22(E)-diene-3beta-ol(4), beta-daucosterol(5), 3-hydroxy-12-taraxasten-28-oic-acid(6) and oleanic acid(7). Compounds 1 (IC50 25.41 mg x L(-1)),3(IC0 4.42 mg L(-1)),4(IC50 11.50 mg x L(-1)),6(IC50 14.17 mg x L(-1)) and 7(IC50 2.88 mg x L(-1)) had higher inhibitory activities than that of acarbose (IC50 1103.01 mg x L(-1)) as the positive control drug.

CONCLUSIONCompound 1-7 were isolated from this plant for the first time. Compound 6 was isolated from Gesneriaceae family for the first time. Compound 7 was isolated from Aeschynanthus genus for the first time.

This study investigated the reversal effect of isotetrandrine, an isoquinoline alkaloid extracted from Caulis mahoniae, on P-glycoprotein-mediated multidrug resistance in human breast cancer doxorubicin-resistant (MCF-7/DOX) cells. RT-PCR assay and immunity histochemistry assay were used to determine the expression level of mdrl gene and P-gp in MCF-7/DOX cells to elucidate resistant character of MCF-7/DOX cells. The activity of isotetrandine to enhance doxorubicin cytotoxicity was tested using MTT (3-(4, 5-dimethyhthiazol)-2,5 -diphenyltetrazolium bromide) assay and was evaluated by the reversal fold (RF) values. Intracellular accumulation of doxorubicin was assessed by the determination of doxorubicin-associated fluorescence intensity. Effect of isotetrandrine on the expression level of P-gp in MCF-7/DOX cells was then determined by immunity histochemistry assay. The ability of isotetrandrine to inhibit P-gp function was evaluated by detecting the accumulation and efflux of rhodamine 123 (Rh123) with flow cytometry (FCM). Verapamil was employed as a comparative agent in whole experiment. The results indicated that MCF-7/DOX cells had phenotype of MDR and that the positive expression of P-gp was their resistant character. 10 microg x mL(-1) isotetrandrine could distinctly enhance cytotoxicity of DOX in MCF-7/DOX cells and reversal fold (RF) was significantly higher than that of verapamil (P < 0.05), but it hardly affected cytotoxicity of DOX in MCF-7 cells and the expression level of P-gp in MCF-7/DOX cells. The ability of isotetrandrine to inhibit P-gp function was reversible, because incubation of MCF-7/DOX cells with isotetrandrine caused a marked increase in uptake and a notable decrease in efflux of Rh123 and a marked increase of intracellular DOX concentrations. In conclusion, isotetrandrine exhibited potent effect on the reversal of P-gp-mediated MDR in vitro, suggesting that it might become a candidate of effective MDR reversing agent in cancer chemotherapy.
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Benzylisoquinolines ; isolation & purification ; pharmacology ; Breast Neoplasms ; pathology ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Gene Expression Regulation, Neoplastic ; Genes, MDR ; Humans ; Mahonia ; chemistry ; Plant Stems ; chemistry ; Plants, Medicinal ; chemistry ; Rhodamine 123 ; metabolism

BACKGROUND: Tumor necrosis factor-associated protein 1 (TRAP1) is a heat-shock protein 90-related mitochondrial chaperone. Accumulative evidence has demonstrated that TRAP1 overexpression is closely related to carcinogenesis. However, the exact function and mechanism of TRAP1 in the occurrence of laryngeal carcinoma remains unclear. OBJECTIVE: To investigate whether RNA interference can inhibit TRAP1 overexpression and to explore its effects on growth and apoptosis of CD133+CD44+ laryngeal carcinoma stem cells. METHODS: CD133+CD44+ laryngeal carcinoma stem cells were sorted from human laryngeal carcinoma Hep-2 cellsusing immunomagnetic beads. The shRNA sequence of TRAP1 was designed and synthesized and CD133+CD44+ laryngeal carcinoma stem cells were transfected with LipofectamineTM 2000. Cell counting kit-8 assay, colony formation assay and flow cytometry were used to investigate the effects of interference of TRAP1 expression on growth and apoptosis of CD133+CD44+ laryngeal carcinoma stem cells. Spectrophotometric method was used to detect the activity of caspase-3, -8 and -9. RESULTS AND CONCLUSION: TRAP1 mRNA and protein expression levels were significantly decreased in TRAP1 shRNA-transfected CD133+CD44+ laryngeal carcinoma stem cells (P < 0.01). Compared with the blank control and negative control groups, the growth and colony formation of CD133+CD44+ laryngeal carcinoma stem cells were significantly inhibited in the TRAP1 shRNA-transfected group (P < 0.05). Apoptosis of CD133+CD44+ laryngeal carcinoma stem cells was significantly inhibited in the TRAP1 shRNA-transfected group as compared with the blank control and negative control groups (P < 0.05). TRAP1 shRNA-mediated cell apoptosis was associated with the activation of caspase-3, -8 and -9. These results suggest that RNA interference targeting inhibition of TRAP1 suppresses cell growth but promotes apoptosis in CD133+CD44+ aryngeal carcinoma stem cells. TRAP1 is likely to be a gene target for treatment of laryngeal carcinoma.

PURPOSE: Hepatitis C virus (HCV) infection is a major cause of liver disease. Several miRNAs have been found to be associated with HCV infection. This study aimed to investigate the functional roles and possible molecular mechanisms of miR-215 in HCV replication. MATERIALS AND METHODS: The expression levels of miR-215 and TRIM22 were detected by quantitative real-time PCR (qRT-PCR) and western blot analysis in Con1b subgenomic genotype 1b HCV replicon cells (Con1b cells) and JFH1 full genome infecting Huh7.5.1 cells (Huh7.5.1 cells). HCV RNA levels were measured by qRT-PCR. The protein levels of NS3, NS5A, p65 subunit of NF-κB (p65), and phosphorylated p65 (p-p65) were determined by western blot analysis. The relationship between miR-215 and TRIM22 were explored by target prediction and luciferase reporter analysis. RESULTS: miR-215 overexpression enhanced HCV replication in Con1b cells, while miR-215 knockdown suppressed HCV replication in Huh7.5.1 cells. TRIM22 was confirmed to be a direct target of miR-215. TRIM22 upregulation resulted in a decline in HCV replication, while TRIM22 inhibition led to enhancement of HCV replication. Additionally, exogenous expression of TRIM22 reversed the facilitating effect of miR-215 on HCV replication, while TRIM22 downregulation counteracted the inhibitory effect of miR-215 knockdown on HCV replication. Furthermore, miR-215 targeted TRIM22 to block the NF-κB pathway, and exerted a positively regulatory role on HCV replication. CONCLUSION: miR-215 facilitated HCV replication via inactivation of the NF-κB pathway by inhibiting TRIM22, providing a novel potential target for HCV infection.
Blotting, Western ; Down-Regulation ; Genome ; Genotype ; Hepacivirus ; Liver Diseases ; Luciferases ; MicroRNAs ; Real-Time Polymerase Chain Reaction ; Replicon ; RNA ; Up-Regulation

Objective: To investigate the effect of hsa_circ_0000392 (circ_0000392) on the radiosensitivity of cervical cancer cells and explore its potential mechanism. Methods: Cervical cancer tissues and adjacent normal tissues of 42 patients with cervical cancer who were confirmed pathologically for the first time in Huaihe Hospital of Henan University from 2016 to 2019 were collected. According to the patients' response to radiotherapy, the cancer tissues were divided into radio-sensitive tissues and radio-resistant tissues. The expressions of circ_0000392, miR-145-5p, and CRKL in radiation-sensitive, radiation-resistant cervical cancer tissues and Hela, SiHa cells were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. SiRNA circ_0000392, miR-145-5p mimic, miR-145-5p inhibitor, pcDNA 3.1-CRKL and its negative control were transfected into HeLa and Siha cells, respectively. After radiation induction, the survival fraction of cells was detected by clone formation assay, apoptosis was detected by flow cytometry, and the expressions of apoptosis-related proteins Bax and Bcl-2 and ERK pathway protein p-ERK1/2 and ERK1/2 were detected by western blot. The targeting relationship between circ_0000392, miR-145-5p and CRKL was verified by dual luciferase reporter gene assay. The effect of circ_0000392 on radiotherapy sensitivity of cervical cancer in vivo was observed in the tumor formation experiment in nude mice. Results: circ_0000392 and CRKL were upregulated in radiation-resistant tissues and cancer cells of cervical cancer, while miR-145-5p was downregulated. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ 6 Gy group were (78.67±10.97) and (71.00±9.54), respectively, which were lower than those in si-Ctrl+ 6 Gy group [(176.00±22.27) and (158.33±17.56), respectively]. The apoptosis rates were (41.55±3.40)% and (31.41±3.29)%, respectively, which were higher than those in si-Ctrl+ 6 Gy group [(15.91±1.37)% and (13.70±1.89)%, P<0.05]. The protein expression of Bax was higher than that of si-Ctrl+ 6 Gy group, and the protein expressions of Bcl2 was lower than those of si-Ctrl+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ miR-145-5p inhibitor+ 6 Gy group were (171.33±25.01) and (137.00±21.66), higher than those in si-circ_0000392#1+ inhibitor NC+ 6 Gy group [(84.67±17.79) vs (71.00±11.00), P<0.05]. The apoptosis rates were (17.41±2.58) % and (15.96±1.25) %, lower than those of si-circ_0000392 #1+ inhibitor NC+ 6 Gy [(40.29±2.92)% and (30.82±2.34)%, respectively, P<0.05]. The expression of Bax protein was lower than that of si-circ_0000392#1+ inhibitor NC+ 6 Gy group, and the expressions of Bcl2 protein were higher than those of si-circ_0000392#1+ inhibitor NC+ 6 Gy group. Circ_0000392 can target miR-145-5p, and CRKL is the downstream target gene of miR-145-5p. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ 6 Gy group were (74.33±10.02) and (66.00±12.17), respectively, which were lower than those of mimic NC+ 6 Gy group [(197.67±17.21) vs (157.67±11.59), respectively, P<0.05]. The apoptosis rates were (45.58±2.16)% and (32.10±3.55)%, higher than those of mimic NC+ 6 Gy group [(15.85±2.45)% and (13.99±1.69)%, respectively, P<0.05]. The expression of Bax protein was higher than that of the mimic NC+ 6 Gy mimic group, and the expression of Bcl2 protein was lower than that of the mimic NC+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ pcDNA-CRKL+ 6 Gy group were (158.00±15.88) and (122.33±13.65), respectively, which were higher than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(71.33±8.02) vs (65.67±12.22), P<0.05]. The apoptosis rates were (19.50±3.45)% and (17.04±0.94)%, respectively, which were lower than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(44.33±2.36)% and (32.05±2.76)%, respectively, P<0.05]. The expression of Bax protein was lower than that of miR-145-5p mimic+ pcDNA group+ 6 Gy group, and the expression of Bcl2 protein was higher than that of miR-145-5p mimic+ pcDNA+ 6 Gy group. Sh-circ_0000392 group had smaller tumor volume and decreased tumor weight (P<0.05). The relative mRNA expression levels of circ_0000392, miR-145-5p and CRKL and the relative protein expression levels of CRKL, Bcl-2 and p-ERK1/2 were decreased, while the relative expression level of Bax protein was increased (P<0.05). Conclusion: Circ_0000392 could enhance the radiosensitivity of cervical cancer cells, and its mechanism may be related to the regulation of CRKL/ERK signaling pathway by targeting miR-145-5p, which provides a new reference for enhancing the radiosensitivity of cervical cancer cells.
Animals ; Mice ; Female ; Humans ; Uterine Cervical Neoplasms/radiotherapy* ; bcl-2-Associated X Protein/genetics* ; Mice, Nude ; Proto-Oncogene Proteins c-bcl-2/genetics* ; Apoptosis ; MicroRNAs/genetics* ; Cell Proliferation ; Cell Line, Tumor

Objective: To investigate the effect of hsa_circ_0000392 (circ_0000392) on the radiosensitivity of cervical cancer cells and explore its potential mechanism. Methods: Cervical cancer tissues and adjacent normal tissues of 42 patients with cervical cancer who were confirmed pathologically for the first time in Huaihe Hospital of Henan University from 2016 to 2019 were collected. According to the patients' response to radiotherapy, the cancer tissues were divided into radio-sensitive tissues and radio-resistant tissues. The expressions of circ_0000392, miR-145-5p, and CRKL in radiation-sensitive, radiation-resistant cervical cancer tissues and Hela, SiHa cells were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. SiRNA circ_0000392, miR-145-5p mimic, miR-145-5p inhibitor, pcDNA 3.1-CRKL and its negative control were transfected into HeLa and Siha cells, respectively. After radiation induction, the survival fraction of cells was detected by clone formation assay, apoptosis was detected by flow cytometry, and the expressions of apoptosis-related proteins Bax and Bcl-2 and ERK pathway protein p-ERK1/2 and ERK1/2 were detected by western blot. The targeting relationship between circ_0000392, miR-145-5p and CRKL was verified by dual luciferase reporter gene assay. The effect of circ_0000392 on radiotherapy sensitivity of cervical cancer in vivo was observed in the tumor formation experiment in nude mice. Results: circ_0000392 and CRKL were upregulated in radiation-resistant tissues and cancer cells of cervical cancer, while miR-145-5p was downregulated. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ 6 Gy group were (78.67±10.97) and (71.00±9.54), respectively, which were lower than those in si-Ctrl+ 6 Gy group [(176.00±22.27) and (158.33±17.56), respectively]. The apoptosis rates were (41.55±3.40)% and (31.41±3.29)%, respectively, which were higher than those in si-Ctrl+ 6 Gy group [(15.91±1.37)% and (13.70±1.89)%, P<0.05]. The protein expression of Bax was higher than that of si-Ctrl+ 6 Gy group, and the protein expressions of Bcl2 was lower than those of si-Ctrl+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ miR-145-5p inhibitor+ 6 Gy group were (171.33±25.01) and (137.00±21.66), higher than those in si-circ_0000392#1+ inhibitor NC+ 6 Gy group [(84.67±17.79) vs (71.00±11.00), P<0.05]. The apoptosis rates were (17.41±2.58) % and (15.96±1.25) %, lower than those of si-circ_0000392 #1+ inhibitor NC+ 6 Gy [(40.29±2.92)% and (30.82±2.34)%, respectively, P<0.05]. The expression of Bax protein was lower than that of si-circ_0000392#1+ inhibitor NC+ 6 Gy group, and the expressions of Bcl2 protein were higher than those of si-circ_0000392#1+ inhibitor NC+ 6 Gy group. Circ_0000392 can target miR-145-5p, and CRKL is the downstream target gene of miR-145-5p. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ 6 Gy group were (74.33±10.02) and (66.00±12.17), respectively, which were lower than those of mimic NC+ 6 Gy group [(197.67±17.21) vs (157.67±11.59), respectively, P<0.05]. The apoptosis rates were (45.58±2.16)% and (32.10±3.55)%, higher than those of mimic NC+ 6 Gy group [(15.85±2.45)% and (13.99±1.69)%, respectively, P<0.05]. The expression of Bax protein was higher than that of the mimic NC+ 6 Gy mimic group, and the expression of Bcl2 protein was lower than that of the mimic NC+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ pcDNA-CRKL+ 6 Gy group were (158.00±15.88) and (122.33±13.65), respectively, which were higher than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(71.33±8.02) vs (65.67±12.22), P<0.05]. The apoptosis rates were (19.50±3.45)% and (17.04±0.94)%, respectively, which were lower than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(44.33±2.36)% and (32.05±2.76)%, respectively, P<0.05]. The expression of Bax protein was lower than that of miR-145-5p mimic+ pcDNA group+ 6 Gy group, and the expression of Bcl2 protein was higher than that of miR-145-5p mimic+ pcDNA+ 6 Gy group. Sh-circ_0000392 group had smaller tumor volume and decreased tumor weight (P<0.05). The relative mRNA expression levels of circ_0000392, miR-145-5p and CRKL and the relative protein expression levels of CRKL, Bcl-2 and p-ERK1/2 were decreased, while the relative expression level of Bax protein was increased (P<0.05). Conclusion: Circ_0000392 could enhance the radiosensitivity of cervical cancer cells, and its mechanism may be related to the regulation of CRKL/ERK signaling pathway by targeting miR-145-5p, which provides a new reference for enhancing the radiosensitivity of cervical cancer cells.
Animals ; Mice ; Female ; Humans ; Uterine Cervical Neoplasms/radiotherapy* ; bcl-2-Associated X Protein/genetics* ; Mice, Nude ; Proto-Oncogene Proteins c-bcl-2/genetics* ; Apoptosis ; MicroRNAs/genetics* ; Cell Proliferation ; Cell Line, Tumor

OBJECTIVETo study the dispersivity of baicalin solid dispersion by Raman spectrometry and infrared spectrometry in order to obtain a new and simple method in examining the dispersivity of solid dispersion.

METHODBaicalin solid dispersion was prepared by solvent method. The Raman spectra, infrared spectra and X-ray powder diffraction patterns of crude baicalin, PVP K30, physical mixture, solid dispersion were acquired by Raman and Fourier transform infrared spectrometers and X-ray powder diffractometry. Then the spectra were compared with each other in order to find out the differences between baicalin solid dispersion and other materials.

RESULTThe result of Raman spectrometry was that amorphous baicalin presented in the solid dispersion, which was in agreement with that of X-ray powder diffractometry.

CONCLUSIONRaman spectrometry which is fast, direct and non-destructive is an ideal method for examining the dispersivity of solid dispersion.