This study examined the association of polymorphisms in angiotensin II receptor genes (AT1R and AT2R) with the risk for aldosterone-producing adenoma (APA) in a Chinese Han population. Four polymorphisms including rs5182 (573T/C) in exon 4, rs5186 (1166A/C) in 3′-untranslated region (3′-UTR) in AT1R gene and rs5194 (2274G/A) in 3′-UTR, rs1403543 (1675G/A) in intron 1 in AT2R gene were detected in 148 APA patients and 192 normal subjects (serving as control) by using a MGB-Taqman probe. The distribution of genotypes of each locus was in accordance with Hardy-Weinberg Equilibrium (HWE) in the APA and control groups (P>0.05). The allele A frequency at rs5194 was significantly higher in the APA group (0.49) than in the control group (0.35) (χ2=12.08, P=0.001). Subjects with homozygotic genotype AA and heterozygotic genotype GA were at an increased risk for APA as compared to those with GG genotype (OR=2.66, 95% CI=1.45–4.87; OR=1.67, 95% CI=1.02–2.74). Furthermore, rs5194 single-nucleotide polymorphism (SNP) at AT2R gene was significantly associated with APA in additive (OR=1.64, 95% CI=1.21–2.20, P=0.001), dominant (OR=1.94, 95% CI=1.23–3.06, P=0.003), and recessive model (OR=2.01, 95% CI=1.17–3.45, P=0.01). It was concluded that rs5194 polymorphism at AT2R gene was associated with the risk for APA, which may constitute a genetic marker of APA.
In order to investigate the influence of silencing soluble epoxide hydrolase (sEH) with double-stranded small interfering RNA (siRNA) on cardiomyocytes apoptosis induced by doxorubicin (DOX), two plasmids containing siRNA sequences specific to sEH were constructed and transfected into the primary cultured cardiomyocytes by using FuGENE HD transfection agents. The mRNA and protein expression levels of sEH were detected by semiquantitative RT-PCR and Western blotting respectively, and the plasmids that silenced sEH most significantly were selected, and renamed EH-R. The plasmids carrying a nonspecific siRNA coding sequence (PCN) served as the negative control. Cardiomyocytes were divided into four groups: control group, DOX group, PCN+DOX group, and EH-R+DOX group. Apoptosis of cardiomyocytes was induced by DOX at a concentration of 1 μmol/L. Apoptosis rate of cardiomyocytes was determined by flow cytometery. The protein expression levels of Bcl-2 and Bax were detected by Western blotting. The results showed that the expression of sEH was down-regulated by EH-R plasmid. The expression levels of sEH mRNA and protein in the EH-R+DOX group were significantly decreased as compared with other groups (P<0.01). As compared with the control group, the apoptosis rate of cardiomyocytes in three DOX-treated groups was obviously increased, the expression levels of Bax increased, and those of Bcl-2 decreased (P<0.01). However, the expression levels of Bax were decreased, those of Bcl-2 increased and the apoptosis rate of cardiomyocytes obviously decreased in EH-R+DOX group when compared with those in the DOX group and the PCN+DOX group (P<0.01 for each). It was concluded that the recombinant plasmids could be successfully constructed, and transfected into the primary cultured cardiomyocytes. They could ameliorate the DOX-induced cardiomyocytes apoptosis by selectively inhibiting the expression of sEH with RNAi and increasing the expression of Bcl-2.
To investigate the protective effects of eplerenone on adriamycin-induced renal injury and the possible mechanisms involved, 36 male Sprague-Dawley rats were randomly divided into control group, adriamycin nephropathy (AN) group and eplerenone-treated group (100 mg·kg−1·d−1 eplerenone). Blood pressure, 24-h urinary protein, serum potassium, sodium and creatinine were measured 28 days after adriamycin injection (a single tail intravenous injection of 6.5 mg/kg adriamycin). The morphological changes of renal tissues were observed by light and electron microscopy. Immunohistochemistry and Western blotting were performed to examine the expression of TGF-β1 and desmin in renal cortex. The results showed that 28 days after adriamycin injection, there were no significant changes in the level of serum potassium, sodium, creatinine concentrations and blood pressure values in the rats of the three groups. Meanwhile, the 24-h proteinuria excretion in the AN group was significantly higher than that in the control group (P<0.01), but that in the eplerenone-treated group was substantially reduced when compared with that in the AN group (P<0.05). Mild mesangial cell proliferation and matrix expansion, diffuse deformation and confluence of foot processes in podocytes were found in the AN group. By contrast, rats in the eplerenone-treated group exhibited obvious attenuation of these morphological lesions. The protein expression of TGF-β1 and desmin in the AN group was markedly up-regulated in contrast to that in the control group (P<0.01), whereas that in the eplerenone-treated group was much lower than in the AN group (P<0.05). It was concluded that eplerenone may ameliorate the proteinuria and the development of pathological alteration in adriamycin-induced nephropathy presumably via the inhibition of cytokine release, and restore the morphology of podocytes independent of its blood pressure-lowing effects.
The present study investigated the enhanced radiosensitivity of U-251 cells induced by sodium butyrate (NaB) and its possible mechanisms. Increased radiosensitivity of U251 cells was examined by clonogenic cell survival assays. The expression of Ku70 mRNA and protein was detected by using RT-PCR and Western blotting respectively. γ-H2AX foci were measured at different time points after ionizing irradiation alone or combined with NaB treatment. The results showed that cell survival rate was significantly reduced, both D0 and Dq values were decreased (D0: 1.43 Gy vs. 1.76 Gy; Dq: 1.22 Gy vs. 2.05 Gy) after the combined treatment as compared with irradiation alone, and sensitivity enhancing ratio (SER) reached 1.23. The average number of γ-H2AX foci per cell receiving the combined treatment was significantly increased at different time points, and the expression levels of Ku70 mRNA and protein were suppressed by NaB in a dose-dependent manner. It was concluded that enhanced radiosensitivity induced by NaB involves an inhibited expression of Ku70 and an increase in γ-H2AX foci, which suggests decreased ability in DSB repair.
Obesity is an established risk factor for endometrial cancer. Leptin, a secreted protein of the ob gene by white adipose tissue, plays an important role in the regulation of food intake and energy consumption in the brain and acts as a potential growth stimulator in normal and neoplastic cancer cells. However, a direct role for leptin in endometrial cancer has not been demonstrated. In the present study, the effect of leptin on the proliferation of Ishikawa endometrial cancer cells was investigated as well as the possible mechanism(s) underlying this action in endometrial cancers which express both short and long isoforms of leptin receptors. The expression of leptin receptor (ObRb) in Ishikawa cells was detected by RT-PCR and Western blotting. The cells after serum starvation, were treated by leptin with various concentrations (0, 10, 50, 100, 150 ng/mL) for different durations (6, 12, 24 h). The effect of leptin treatment on cell proliferation was examined by MTT assay. Meanwhile, inhibitory effect of Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) inhibitor AG490 or extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 on the proliferation of Ishikawa cells induced by leptin was also studied. Ishikawa cells were treated with 100 ng/mL leptin for various periods (0, 20, 40, 60 min), and the levels of STAT3 phosphorylation and ERK1/2 phosphorylation were examined by Western blotting. The results showed that leptin induced the phosphorylation of STAT3 and the activation of ERK1/2 in a time- and dose-dependent manner in the Ishikawa endometrial cancer cells. Blocking STAT3 phosphorylation with the inhibitor AG490, or blocking ERK1/2 activation by the specific ERK1/2 kinase inhibitor, PD98059, abolished leptin-induced proliferation of Ishikawa cells. In addition, leptin was found to potently induce the invasion of endometrial cancer cells in a Matrigel invasion assay. Leptin-stimulated invasion was effectively blocked by pharmacological inhibitors of STAT3 (AG490) and ERK1/2 kinase (PD98059). These results suggested that leptin promotes endometrial cancer growth and invasiveness by activating STAT3 and ERK1/2 signaling pathways and therefore blocking its action at the receptor level can be a rational therapeutic strategy.
This study examined the impacts of intrauterine murine cytomegalovirus (MCMV) infection on the long-term learning and memory of offspring. Sexually matured male and female BALB/C mice without MCMV infection were identified by ELISA and then mated. Seventy pregnant mice were randomly divided into the virus group (n=40) and the control group (n=30), in which the pregnant mice were subjected to placenta inoculation of MCMV suspension (1 μL, 1×106 PFU) or the same amount of cell culture medium, respectively, at gestational age of 12.5 days. Some pregnant mice [virus group (n=20), control group (n=15)] were sacrificed by cervical dislocation at gestational age of 18.5 days, and the head circumference and brain weight of the mouse fetuses were measured, and the MCMV infection in their brain tissues was detected by PCR. The other pregnant mice [virus group (n=20), control group (n=15)] delivered naturally, and the learning and memory capability of the offspring at 70-day-old was analyzed by Morris water maze test. The results showed that 28.57% mouse fetuses in the virus group developed viral infection in the brain. Their head circumference and brain weight were significantly reduced as compared with those in the control group (P<0.01). The Morris water maze test revealed that the mouse offspring in the control group found the platform with straight-line trajectories after training. In contrast, the counterparts in the virus group intended to enter the central area, but looked for the platform with a circular trajectory. And the infected mice exhibited prolonged swimming distance and swimming latency (P<0.01). It was concluded that: (1) placenta inoculation of MCMV can cause fetal brain infection and intrauterine development retardation; (2) the offspring of MCMV placenta inoculation mice showed a long-term decline in learning and memory capability.
This study examined the analgesic effect of diprospan in rats with trigeminal neuralgia. Rat model of trigeminal neuralgic pain was established by loosely ligating the left infraorbital branch of the trigeminal nerve. After allodynia developed, the rats were randomly divided into 2 groups (n=20 in each): diprospan group, in which the rats received diprospan (7 mg/mL, 0.1 mL) injected to the left infraorbital foramen area; control group, in which saline (0.1 mL) was administered as the same manner as the diprospan group. The pain threshold (PT) in the left infraorbital area was measured before and 2, 6, and 8 weeks after the administration. The expression of neuropeptides [substance P, preprotachykinin A (PPTA), calcitonin gene-related peptide (CGRP)] in the trigeminal nerve was detected at the same time points as the PT measurement by immunohistochemistry or in situ hybridization method. The results showed that in the diprospan group, the PT was 10.65±1.26, 10.77±1.19 and 14.13±1.34 g 2, 6, and 8 weeks after the administration respectively, significantly higher than that before the administration (PT value: 0.36±0.11) (P<0.05 for each). In the saline group, the PT was 0.37±0.13, 0.66±0.09, 4.45±1.29 and 13.72±1.72 g before and 2, 6, and 8 weeks after the administration respectively with differences being significant between before and 6, 8 weeks after the administration (P<0.01). No significant difference existed in the PT between the diprospan group and the saline group at pre-administration (P>0.05). The PT in the diprospan group was significantly greater than that in the saline group 2 and 6 weeks post-administration (P<0.05). In the diprospan group, the expression levels of neuropeptides were significantly reduced as compared with those in the saline group 2 and 6 weeks post-administration (P<0.05). It was concluded that diprospan has an obvious analgesic effect on the trigeminal neuropathic pain partly by reducing the expression of neuropeptides in the trigeminal ganglia.
This study aimed to modify the mixed and purified culture of rat retinal ganglion cells (RGCs) in vitro. The retinae of 1–3 day old Sprague-Dawley (SD) rats were separated bluntly into two layers: inner layer and outer layer, under a surgical microscope. Retinal cells isolated from different layers (inner layer, outer layer and whole retinal tissue) by using enzyme dissociation method were cultured in F12/DMEM medium containing 15% FBS. After 3-day culture, the RGCs in the retinal cells obtained from mixed culture of inner, outer, and whole retinal tissue were identified by immunocytochemical staining of Thy-1.1, and the rate of RGCs to retinal cells (RGCs%) was calculated. Two monoclonal antibodies, anti-macrophages/granulocytes (OX-41) against rat macrophage and antibody against rat Thy-1.1 (OX-7), were used to purify RGCs by either a conventional or modified two-stepped immunopanning procedure (purification in situ). Purified RGCs were seeded at different cell density and cultured in F12/DMEM medium containing 15% FBS. Immunocytochemical staining for Thy-1.1, MTT, and PI-Hoechst33342 fluorescence imaging were used to identify the purity and the viability of RGCs in purified culture of RGCs. The results showed: (1) Immunocytochemistry of different retinal tissue layers culture revealed that the RGCs% was (19.9±1.2)%, (0.5±0.2)%, and (6.2±1.7)% respectively in the mixed culture of inner, outer, and whole retinal tissue, with differences being significant (P<0.05); (2) fluorescent double staining of Hoechst33342 and PI indicated that with the same RGCs%, RGCs obtained from purification in situ grew well with more neurite outgrowth than those by the conventional two-stepped immunopanning method; (3) the viability of purified RGCs seeded at high density was increased and the cells developed complex intercellular networks. The viability of RGCs was declined with the decreasing seeding density, and most cells presented round or oval in shape with thin neurites. It was concluded that: (1) RGCs% in the inner layer retina was higher than that in the outer layer retina; (2) RGCs obtained by in situ purification had more neurite outgrowth and lower mortality than those by conventional two-stepped immunopanning procedure; (3) the viability of purified RGCs could be increased by increasing cell seeding density to some extent.
This study examined the clinical features of complications of congenital retinoschisis and the clinical efficacy of vitreoretinal surgery in the treatment of these complications. The clinical efficacy of surgical treatments was retrospectively analyzed in 10 patients with congenital retinoschisis (10 eyes) complicated with rhegmatogenous retinal detachment (n=5), vitreous hemorrhage (n=2) and macula- involving schisis (n=1). All the patients suffered foveal and peripheral schisis. They were treated with scleral buckling (n=1) or vitrectomy (n=9). After the surgical treatment, the retina was reattached in patients with rhegmatogenous retinal detachment; the refractive media became transparent in those with vitreous hemorrhage; the visual acuity in 80% of patients was improved; no remarkable progression of schisis was found; no severe operative complications occurred. It was concluded that vitreoretinal surgery in the treatment of complications of congenital retinoschisis is safe and effective, and helps improve and maintain the visual function.
This study examined the dynamic characteristics of upper airway collapse at soft palate level in patients with obstructive sleep apnea/hypopnea syndrome (OSAHS) by using dynamic 3-Dimensional (3-D) CT imaging. A total of 41 male patients who presented with 2 of the following symptoms, i.e., daytime sleepiness and fatigue, frequent snoring, and apnea with witness, were diagnosed as having OSAHS. They underwent full-night polysomnography and then dynamic 3-D CT imaging of the upper airway during quiet breathing and in Muller’s maneuver. The soft palate length (SPL), the minimal cross-sectional area of the retropalatal region (mXSA-RP), and the vertical distance from the hard palate to the upper posterior part of the hyoid (hhL) were compared between the two breathing states. These parameters, together with hard palate length (HPL), were also compared between mild/moderate and severe OSAHS groups. Association of these parameters with the severity of OSAHS [as reflected by apnea hypopnea index (AHI) and the lowest saturation of blood oxygen (LSaO2)] was examined. The results showed that 31 patients had severe OSAHS, and 10 mild/moderate OSAHS. All the patients had airway obstruction at soft palate level. mXSA-RP was significantly decreased and SPL remarkably increased during Muller’s maneuver as compared with the quiet breathing state. There were no significant differences in these airway parameters (except the position of the hyoid bone) between severe and mild/moderate OSAHS groups. And no significant correlation between these airway parameters and the severity of OSAHS was found. The position of hyoid was lower in the severe OSAHS group than in the mild/moderate OSAHS group. The patients in group with body mass index (BMI)≥26 had higher collapse ratio of mXSA-RP, greater neck circumference and smaller mXSA-RP in the Muller’s maneuver than those in group with BMI<26 (P<0.05 for all). It was concluded that dynamic 3-D CT imaging could dynamically show the upper airway changes at soft palate level in OSAHS patients. All the OSAHS patients had airway obstruction of various degrees at soft palate level. But no correlation was observed between the airway change at soft palate level and the severity of OSAHS. The patients in group with BMI≥26 were more likely to develop airway obstruction at soft palate level than those with BMI<26.
Murine cytomegalovirus (MCMV) IE3 protein is a multifunctional viral protein that interacts with several target proteins of both viral and host cellular origin. To investigate the biological function of IE3 in the pathogenesis of the brain disorders caused by CMV, a screening for host cellular proteins that could interact with IE3 was performed. By yeast two-hybrid screening, ankyrin repeats domain 17 (Ankrd17, also known as Gtar) was identified as a host factor that could interact with IE3. This interaction was verified by yeast two-hybrid assay and chemiluminescent co-immunoprecipitaion. Mapping analysis suggested that the 1–148 residues of IE3 were responsible for the interaction. These results suggested that the interaction between Ankrd17 and IE3 may play a key role in the pathogenesis of MCMV-associated disease.
This study investigated the conjoined cellular oxidative damage of human embryo kidney 293T (HEK293T) cells induced by cadmium chloride (CdCl2) and nanometer titanium dioxide (nano-TiO2). RT-PCR technique was used to detect the expressions of Heme oxygenase-1 (HO-1) and 8-oxoguanine DNA glycosylase (OGG1). The activities of superoxide dismutase (SOD) and catalase enzyme (CAT) and concentrations of reactive oxygen species (ROS) and maldondialdehyde (MDA) were measured by different approaches. The results showed that CdCl2 and nano-TiO2 at a low concentration of 0.75 total toxic unit (TU) exerted an additive effects on HO-1 gene expression, CAT activities and MDA concentrations. When the total TU was increased to 1 or 1.25 TU, the interaction was synergetic. Moreover, the mixture with high proportion of CdCl2 produced an additive effect on the OGG1 gene expression, and the interaction was changed to be synergetic when the concentration of CdCl2 was lower than or equal to that of nano-TiO2. Synergetic effects of CdCl2 and nano-TiO2 on cellular oxidative damage of HEK293T cells were found as indicated by the changes in the SOD activities and ROS concentrations. It was concluded that CdCl2 and nano-TiO2 exerts synergistic effects on the cellular oxidative damage of HEK293T cells, and the sensitivity of these indicators of oxidative damage varies with the proportion of CdCl2 and nano-TiO2 in the mixture.
This study examined the association of a common polymorphic allele (25G) of the low-density lipoprotein receptor-related protein1 (LRP1) gene with myocardial infarction (MI). The genotypes of LRP1 25CG (rs35282763) were determined in 347 MI patients and 347 age- and sex-frequency-matched controls from an unrelated Chinese Han population. Factor VIII (FVIII) levels were measured in the MI patients and controls by chromogenic assay and enzyme-linked immunosorbent assay (ELISA). The results showed that LRP1 25CG (rs35282763) genotype distribution did not differ significantly between patients (n=206 for 25CC, n=122 for 25CG) and controls (n=191 for 25CC, n=126 for 25CG; P>0.05). The 25G allele was not associated with a reduced risk of MI (P>0.05). Further stratifications for age, sex, and other cardiovascular risk factors did not affect the negative findings. It was concluded that the presence of the G allele at the 25CG (rs35282763) polymorphism of the LRP1 is not associated with a reduced risk of MI, and genotyping for LRP1 25CG (rs35282763) polymorphism is not useful in assessing the individual risk of MI.
SUMO4 Met55Val variation was shown to be related to type 2 diabetes susceptibility and the vascular complications in Asian people. To further examine the related mechanisms, this study was designed to evaluate the association of SUMO4 Met55Val polymorphism with insulin resistance and β cell function in newly diagnosed type 2 diabetic patients in a Chinese population. Four hundred and twenty seven newly diagnosed type 2 diabetic patients were selected for SUMO4 Met55Val polymorphism genotype analysis. All subjects underwent a 75-g oral glucose tolerance test (OGTT) to estimate the insulin sensitivity and β cell function. Anthropometrics and a metabolic profile were used for phenotyping analysis. The results showed that the SUMO4 Met55Val polymorphism was associated with higher insulin resistance (P<0.001) and lower insulin sensitivity (P<0.001). Patients with GG genotype had higher levels of plasma glucose, insulin and C peptide. Insulin sensitivity index (ISI) was closely correlated with body mass index (BMI) in patients with GG genotype in comparison to the counterparts with AG or AA genotype (r= −0.504 vs. r= −0.430 vs. r= −0.340). Multiple regression linear analysis showed that SUMO4 Met55Val polymorphism was an independent determinant for insulin sensitivity (P=0.001), which, along with triglyceride, BMI and sex, could account for 20.1% of the variation in ISI. The result remained the same after adjusting for BMI and sex. No association was found between SUMO4 Met55Val polymorphism and β cell function (all P>0.05). It was concluded that SUMO4 Met55Val variant was associated with increased insulin resistance in Chinese patients with newly diagnosed type 2 diabetes.
This study investigated the variation of serum monocyte chemoattractant protein-1 (MCP-1) in patients with both diabetes mellitus (DM) and metabolic syndrome (MS). Based on the International Diabetes Federation (IDF) diagnostic criteria, 93 patients enrolled in this study were divided into four groups: normal control (NC), simple DM, simple MS, and DM plus MS (DM-MS) groups. The main measures included height, weight, waist circumference (WC), hip circumference, blood pressure, fasting blood glucose, insulin resistance index (HOMA-IR), serum triglyceride (TG), HDL-ch, LDL-ch, and MCP-1. The results showed that the serum levels of MCP-1 in the DM-MS group were significantly increased as compared with those in the DM and MS groups (P<0.05), and the increase in the MCP-1 level in the DM group was much higher than in the MS group (P<0.05). The DM-MS group had the highest HOMA-IR levels, followed by MS, DM and NC groups (P<0.05). Correlation tests showed that the association of MCP-1 with age, HDL-ch, or LDL-ch was insignificant, whereas that of MCP-1 with body mass index (BMI), waist hip rate (WHR), WC, systolic blood pressure (SBP), diastolic blood pressure (DBP), TG, and HOMA-IR was significantly positive. It was concluded that circulating MCP-1 was substantially increased in patients with both DM and MS as compared with that in the patients with DM or MS alone, and the central obese state may contribute to a more vicious proinflammatory condition and insulin resistance in patients with diabetes.
This study investigated the effects of telmisartan on insulin resistance in high-fat diet-treated rats and the possible mechanism. A total of 40 male Sprague-Dawley rats enrolled in the study were divided into 4 groups at random: ND group (n=10) and HD group (n=10), in which the rats were given a normal chow diet or a high-fat diet for 20 weeks following a one-week adaptation; ND+telmisartan (n=10) group and HD+telmisartan group (n=10), in which the rats were initially administered in the same way as the ND or HD group, and then they were orally gavaged with telmisartan (5 mg/kg daily) additionally for 5 weeks. Related inflammatory factors were measured by ELISA. Monocyte chemotactic protein 1 (MCP-1), phosphorylated JNK and IκB-α expressions in both adipose and liver were detected by Western blotting. CRP and angiotensin II receptor 1 (AT1) mRNA expressions in both adipose and liver were determined by RT-PCR. The results showed that telmisartan administration in vivo reversed insulin resistance as evidenced by a decrease in plasma fasting glucose levels, plasma fasting insulin levels and homeostasis model of assessment-insulin resistance (HOMA-IR). Furthermore, telmisartan administration significantly reduced serum CRP, TNF-α and IL-1β levels, and elevated serum IL-10 levels. It was also found to hamper the high-fat diet-induced increase in CRP mRNA, AT1 mRNA and MCP-1, and decrease in IκB-α in both adipose and liver. It was concluded that telmisartan administration in vivo may improve insulin resistance through attenuated inflammatory response pathways.
This study investigated the role of reactive oxygen species (ROS) in the pathogenesis of triptolide-induced renal injury in vivo. Rats were randomly divided into 4 groups (n=5 in each): triptolide group in which the rats were intraperitoneally injected with triptolide solution at a dose of 1 mg/kg of body weight on day 8; control group in which the rats received a single intraperitoneal injection of 0.9% physiological saline on day 8; vitamin C group in which the rats were pretreated with vitamin C by gavage at a dose of 250 mg/kg of body weight per day for 7 days before the same treatment as the control group on day 8; triptolide+vitamin C group in which the rats were first subjected to an oral administration of vitamin C at a dose of 250 mg/kg of body weight per day for 7 days, and then to the same treatment as the triptolide group on day 8. All the rats were sacrificed on day 10. Blood samples were collected for detection of plasma creatinine (Pcr) and plasma urea nitrogen (PUN) concentrations. Both kidneys were removed. The histological changes were measured by haematoxylin-eosin (HE) staining. The production of ROS was determined by detecting the fluorescent intensity of the oxidation-sensitive probe rhodamine 123 in renal tissue. Renal malondialdehyde (MDA) content was measured to evaluate lipid peroxidation level in renal tissue. TUNEL staining was performed to assess apoptosis of renal tubular cells. Renal expression of apoptosis-related proteins Bcl-2, Bax, Bid, Bad, Fas and FasL, as well as corresponding encoding genes were assessed by Western Blotting and real-time PCR. The results showed that triptolide treatment promoted the generation of a great amount of ROS, up-regulated the expression of Bax, Bid, Bad, Fas and FasL at both protein and mRNA levels, as well as the ratio of Bax to Bcl-2, and caused the apoptosis of renal tubular cells and renal injury. However, pretreatment with an antioxidant, vitamin C, significantly reduced the generation of ROS and effectively inhibited the triptolide-induced apoptosis of renal tubular cells and renal injury. It was concluded that ROS plays a critical role in triptolide-induced apoptosis of renal tubular cells and renal injury. The protective administration of vitamin C may help alleviate triptolide-induced renal injury and nephrotoxicity.
This study examined the role of regulated upon activation normal T cell expressed and secreted (RANTES) and its receptor C-C chemokine receptor type 5 (CCR5) in gastric cancer metastasis and the associated mechanism. The expression of RANTES and CCR5 was detected by using immunohistochemical staining and Western blotting in the gastric cancer tissues obtained from 60 gastric cancer patients with or without lymph node metastasis (n=30 in each). The results showed that the expression levels of RANTES and CCR5 were higher in gastric cancer with lymph node metastasis than in that without metastasis (P<0.05). The expression levels of RANTES in 30 lymph nodes with cancerous invasion were higher than in 30 normal lymph nodes (P<0.05). Chemotactic test revealed that the number of migrating gastric cancer cells (n=295.0±54.6) induced by the protein of cancer-invading lymph nodes was greater than that by the protein mixture from cancer-invading lymph nodes and RANTES antibody (n=42.5±11.6) (P<0.05). RT-PCR showed that the expression levels of the main Th1 cytokines (IL-2, Γ-IFN) were lower in gastric cancer with lymph node metastasis (2.22±0.90, 3.26±1.15 respectively) than in that without metastasis (3.07±1.67, 4.77±1.52 respectively) (P<0.05), but the expression level of the main Th 2 cytokine (IL-10) was higher in gastric cancer with lymph nodes metastasis (6.06±2.04) than in that without metastasis (4.88±1.87) (P<0.05). It was concluded that RANTES and its receptor CCR5 may contribute to gastric cancer metastasis through influencing the balance of Th1/Th2. RANTES and CCR5 may become a marker of gastric cancer metastasis.
The effects of targeted silencing of heparanase gene by small interfering RNA (siRNA) on invasiveness and metastasis of osteosarcoma cells (MG63 cells) were investigated in the present study. Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene. The expression vector containing short hairpin RNA (pGenesil-shRNA) was constructed successfully. MG63 cells were randomly allocated into 3 groups: blank group, empty vector (pGenesil) transfected group and expression vector (pGenesil-shRNA) transfected group. Under the induction of Lipofectamine 2000, the recombinants were transfected into MG63 cells. Heparanase gene expression level was detected by RT-PCR and Western blotting. Cell proliferation was measured by MTT assay. Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays. HUVECs migration assay was applied for the detection of angiogenesis. As compared with negative controls, the mRNA and protein expression levels of heparanase were down-regulated by 76.1% (P<0.01) and 75.3% (P<0.01) respectively in the pGenesil-shRNA transfected group. Meanwhile, the proliferation, adhesiveness, invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited. It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.
This study examined the effect of small interfering RNA-mediated β-catenin knockdown on the survival, invasion and chemosensitivity of human osteosarcoma cells (U2-OS cells). The siRNA against β-catenin was constructed and transfected into U2-OS cells. The expression of β-catenin was detected by qRT-PCR and Western blotting. Cell growth and apoptosis was detected in the presence or absence of doxorubicin by MTT and flow cytometry, respectively. Cell invasion ability was measured by transwell assay. The results showed that the transfection of β-catenin siRNA resulted in decreased expression of β-catenin, suppression of invasion and motility of U2-OS cells, reduced chemosensitivity to doxorubicin in vitro, and little change in cell growth and apoptosis. Additionally, down-regulated MT1-MMP expression was found after transfection. It was concluded that knockdown of β-catenin gene may decrease the invasive ability of human osteosarcoma cells through down-regulated MT1-MMP expression, and the chemosensitivity of osteosarcoma cells against doxorubicin.
This study examined the possible mechanism of sublingual immunotherapy (SLIT) in the treatment of allergic asthma. Forty asthma patients allergic to dust mite were enrolled. They received SLIT with dermatophagoides farinae (Der. f) drops for one year. Thirty healthy subjects served as controls. The levels of IL-4 and IFN-γ of peripheral blood mononuclear cells (PBMCs) were determined in allergic asthma patients before and after the SLIT as well as the healthy subjects. The results showed that the level of IL-4 was substantially increased and that of IFN-γ remarkably decreased in the patients before the SLIT as compared with those in the healthy subjects (P<0.05). After the SLIT, the level of IL-4 was significantly reduced and that of IFN-γ elevated in these allergic asthma patients. It was concluded that sublingual immunotherapy is effective for patients with allergic asthma. And it may work by regulating the balance of Th1/Th2 through changing the expression of IL-4 and IFN-γ in PBMCs.
The effects of Cinnamon granules on pharmacokinetics of berberine in Rhizoma Coptidis granules in healthy male volunteers, and the compatibility mechanism of Jiao-Tai-Wan (JTW) composed of Rhizoma Coptidis granules and Cinnamon granules were investigated. The concentration of berberine in plasma of healthy male volunteers was determined directly by high performance liquid chromatography (HPLC) after an oral administration of Rhizoma Coptidis granules alone or combined with Cinnamon granules (JTW). The plasma concentration-time curves of berberine were plotted. The data were analyzed with Drug and Statistics (DAS) 2.0 pharmacokinetic program (Chinese Pharmacology Society) to obtain the main pharmacokinetic parameters. The results showed that the plasma concentration-time curve of berberine was described by a two-compartment model. The Cmax, Tmax, t1/2 and CLz/F of berberine in Rhizoma Coptidis granules were 360.883 μg/L, 2.0 h, 3.882 h, 119.320 L·h−1·kg−1 respectively, and those of berberine in JTW were 396.124 μg/L, 1.5 h, 4.727 h, 57.709 L·h−1·kg−1 respectively. It was suggested that Rhizoma Coptidis granules combined with Cinnamon granules could increase the plasma concentration of berberine, promote berberine absorption and lengthen the detention time of berberine in healthy male volunteers.
Methylmalonic aciduria (MMA) is a common inherited autosomal recessive disorder resulting from defects in the enzyme methylmalonyl CoA mutase (MCM, mut complementation group) or in the synthesis of the MCM cofactor adenosylcobalamin (cbl complementation groups). The defects in the mut complementation group accounts for the largest number of patients with isolated MMA. At least 200 mutations in the MUT gene on chromosome 6p12 have been identified in MMA patients until now. This study aimed to investigate the clinical characteristics of MMA and genomic variations in the MUT gene of Chinese patients. Genomic DNA was extracted from 18 patients who were diagnosed as having isolated MMA by gas chromatography/mass spectrometry (GC-MS), and from some of their parents as well. Amplification and direct sequencing of the MUT coding regions (exon 2–13) and their adjacent intronic consensus splice sites were performed in order to identify the disease causing mutations. In this group, six novel mutations in the MUT gene, c.424A>G (p.T142A), c.786T>G (p.S262R), c.808G>C (p.G270R), c.1323_1324insA, c.1445-1G>A and c.1676+77A>C were identified. p.T142A and p.G270R were respectively detected at a heterozygous level in one patient. Two previously reported mutations, c.682C>T (p.R228X) and c.323G>A (p.R108H) were also found in this study. In addition, six previously described single nucleotide polymorphism (SNP), c.636A>G (p.K212K), c.1495G>A (p.A499T), c.1595A>G (p.H532R), c.1992G>A (p.A664A), c.2011G>A (p.V671I) and c.1677-53A>G were identified. In this study, we updated the spectrum of MUT mutations and identified the main MMA-causing mutations in Chinese MMA patients.
The present study evaluated the effect of non-thermal plasma on skin wound healing in BalB/c mice. Two 6-mm wounds along the both sides of the spine were created on the back of each mouse (n=80) by using a punch biopsy. The mice were assigned randomly into two groups, with 40 animals in each group: a non-thermal plasma group in which the mice were treated with the non-thermal plasma; a control group in which the mice were left to heal naturally. Wound healing was evaluated on postoperative days (POD) 4, 7, 10 and 14 (n=5 per group in each POD) by percentage of wound closure. The mice was euthanized on POD 1, 4, 7, 10, 14, 21, 28 and 35 (n=1 in each POD). The wounds were removed, routinely fixed, paraffin-embedded, sectioned and HE-stained. A modified scoring system was used to evaluate the wounds. The results showed that acute inflammation peaked on POD 4 in non-thermal plasma group, earlier than in control group in which acute inflammation reached a peak on POD 7, and the acute inflammation scores were much lower in non-thermal group than in control group on POD 7 (P<0.05). The amount of granular tissue was greater on POD 4 and 7 in non-thermal group than in control group (P<0.05). The re-epithelialization score and the neovasularization score were increased significantly in non-thermal group when compared with control group on POD 7 and 10 (P<0.05 for all). The count of bacterial colonies was 103 CFU/mL on POD 4 and <20 CFU/mL on POD 7, significantly lower than that in control group (109 CFU/mL on POD 4 and >1012 CFU/mL on the POD 7) (P<0.05). It was suggested that the non-thermal plasma facilitates the wound healing by suppressing bacterial colonization.
In this study, the colonization and distribution of Helicobacter pylori (Hp) in patients with chronic gastric diseases were investigated and the relationship between the periodontal initial treatment and presence of Hp in oral cavity was examined to better understand the connection between Hp infection and chronic diseases. Primers for PCR amplification were designed according to ureC gene and cagA genes of Hp. Specimens were harvested from different sites of 96 patients with chronic gastric diseases and the specimens of dental plaques, gargles and dorsal mucosa were tested for Hp. The 96 patients were treated by bismuth triple therapy and among them, 52 subjects were additionally given periodontal initial therapy. The eradication rate of gastric Hp and oral Hp detection rate were determined 4 weeks and 1 year after the treatment. The results showed that the detection rates of oral specimens were in the order of dental plaques (82.3%), gargles (51.1%) and scrapings of dorsal mucosa of tongue (37.5%). One year after bismuth triple therapy or the triple therapy in combination with periodontal initial treatment, the eradication rate of gastric Hp was significantly higher in the combination treatment group than in group treated by the triple therapy alone (62.8% vs. 32.4%, P<0. 05). Moreover, the Hp detection rate was significantly lower in the combination group than in the group treated only with the triple therapy. We are led to conclude that Hp is present at various parts of oral cavity, oral Hp might be an important source of gastric Hp and the triple therapy plus periodontal initial treatment can enhance the long-term eradication rate of gastric Hp in patient with both chronic gastric diseases and chronic periodontitis.