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ObjectiveTo investigate the effect of Yishen Tongluo prescription （YSTLP） on apoptosis of renal tubular epithelial cells and explore the mechanism based on endoplasmic reticulum stress pathway of protein kinase R-like endoplasmic reticulum kinase （PERK）/activating transcription factor 4 （ATF4）/transcription factor C/EBP homologous protein （CHOP）. MethodThe db/db mice were randomly divided into model group， valsartan group （10 mg·kg-1）， and low， middle， high-dose YSTLP groups （1， 2.5， 5 g·kg-1）. Samples were collected after eight weeks of drug intervention. In addition， db/m mice in the same litter served as the control group. Human renal tubular epithelial cells （HK-2） were cultured in vitro and divided into the control group， advanced glycated end-product （AGE） group， and AGE + low， middle， and high-dose YSTLP groups （100， 200， 400 mg·L-1）. TdT-mediated dUTP nick end labeling （TUNEL） staining was used to detect the apoptosis rate of HK-2 cells. Methylthiazolyldiphenyl-tetrazolium bromide （MTT） assay was conducted to detect the viability of HK-2 cells. Calcium fluorescence probe staining and luciferase reporter gene method were adopted to detect the luciferase activity of folded protein response element （UPRE） and endoplasmic reticulum stress. Immunohistochemical （IHC） analysis was carried out to measure the protein expressions of phosphorylated PKR （p-PERK）， CHOP， and ATF4. Real-time polymerase chain reaction （Real-time PCR） was used to measure the mRNA expression levels of CHOP and X-box binding protein 1 （XBP1） in mouse kidney and HK-2 cells. Western blot was used to detect the protein expression level of p-PERK， PERK， CHOP， ATF4， B-cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， and cleaved Caspase-3 in mouse kidney and HK-2 cells. ResultIn the cellular assay， HK-2 cell viability was significantly reduced， and the apoptosis rate was elevated in the AGE group compared with the control group （P<0.01）. The mRNA and protein expression levels of apoptosis-related factor Bcl-2 were significantly reduced （P<0.01）， and those of Bax were significantly increased （P<0.01）. The protein expression level of cleaved Caspase-3 was significantly increased （P<0.01）. Compared with the AGE group， YSTLP administration treatment resulted in elevated cell viability and reduced apoptosis rate （P<0.01）. The mRNA and protein expression levels of Bcl-2 were significantly elevated in a time- and dose-dependent manner （P<0.01）， and those of Bax were significantly reduced in a time- and dose-dependent manner. The protein expression level of cleaved Caspase-3 was significantly reduced in a time- and dose-dependent manner （P<0.01）. The intracellular Ca2+ imbalance and UPRE luciferase fluorescence intensity were increased in the AGE group compared with the control group （P<0.01）. The mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 were significantly increased （P<0.01）， and the protein expression levels of p-PERK， CHOP， and ATF4 were significantly increased （P<0.05）. Compared with the AGE group， YSTLP effectively improved intracellular Ca2+ imbalance in HK-2 cells and decreased UPRE luciferase fluorescence intensity in a dose-dependent manner （P<0.01）. It reduced the mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 （P<0.01） and the protein expression levels of intracellular p-PERK， CHOP， and ATF4 in a dose- and time-dependent manner （P<0.01）. In animal experiments， the protein expression level of Bcl-2 was significantly reduced（P<0.01）， and that of cleaved Caspase-3 and Bax was significantly increased in the model group compared with the control group （P<0.05）. The protein expression level of Bcl-2 was dose-dependently elevated， and that of cleaved Caspase-3 and Bax was dose-dependently decreased in the YSTLP groups compared with the model group （P<0.01）. Compared with the control group， the mRNA expression levels of CHOP and XBP1 were significantly elevated in the model group （P<0.05， P<0.01）， and the protein expression levels of p-PERK， CHOP， and ATF4 were significantly increased （P<0.05）. Compared with the model group， YSTLP significantly decreased the mRNA expression levels of CHOP and XBP1 （P<0.01） and the protein expression levels of p-PERK， CHOP， and ATF4 （P<0.01）. ConclusionYSTLP can effectively inhibit endoplasmic reticulum stress and improve apoptosis of renal tubular epithelial cells， and its mechanism may be related to the regulation of the PERK/AFT4/CHOP pathway.
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ObjectiveTo establish a mouse model of basilar artery dolichoectasia （BAD） and explore the mechanism of modified Tongqiao Huoxuetang （JTQHX） in regulating BAD via phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） pathway. MethodSixty C57/BL6 female mice were randomized into sham operation （injected with 10 U·mL-1 inactivate elastase）， model， atorvastatin calcium tablets （2.6 mg·kg·d-1）， and low- and high-dose （crude drug 3.4， 17 g·kg-1·d-1， respectively） JTQHX groups. The mouse model of BAD was established by injection with 10 U·mL-1 elastase. After 14 days of modeling， the sham operation group and model group were administrated with equal volumes of pure water by gavage， and other groups with corresponding drugs for 2 months. The levels of interleukin-6 （IL-6） and calpain （LpA） in the serum were measured by enzyme-linked immunosorbent assay （ELISA）. Verhoeff 's Van Gieson （EVG） staining was employed to observe the pathological changes of blood vessels. Terminal-deoxynucleotidyl transferase mediated nick end labeling （TUNEL） was employed to examine the apoptosis rate of vascular smooth muscle cells （VSMCs）. Image Pro Plus was used to observe and calculate the curvature index， elongation length， percentage increase in vessel diameter， and curvature angle of the basilar artery vessels in mice. Western blot was employed to determine the expression levels of PI3K and Akt in the vascular tissue. ResultCompared with the sham operation group， the model group showed lowered IL-6 level （P<0.01）， no significant change in LpA level， increased apoptosis of VSMCs （P<0.01）， and increased curvature index， elongation length， percentage increase in vessel diameter， and curvature angle （P<0.01）. Furthermore， the modeling up-regulated the protein levels of PI3K and Akt in blood vessels （P<0.01） and aggravated the destruction of the inner elastic layer， atrophy of the muscular layer， and hyaline changes in the connective tissue of the medial membrane of the basilar artery wall. Compared with the model group， 2 months of treatment with JTQHX elevated the IL-6 level （P<0.01）， reduced the apoptosis of VSMCs （P<0.01）， decreased the curvature index， elongation length， percentage increase in vessel diameter， and curvature angle （P<0.05， P<0.01）， and down-regulated the protein levels of PI3K and Akt in blood vessels （P<0.01）. In addition， the treatment alleviated the destruction of the inner elastic layer， atrophy of the muscular layer， and hyaline changes in the connective tissue of the medial membrane of the basilar artery wall. ConclusionJTQHX inhibits the elongation， expansion， and curvature of basilar artery vessels and alleviates the pathological changes by reducing the apoptosis of VSMCs and down-regulating the expression of PI3K/Akt pathway.
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OBJECTIVE To explore the effect mechanism of ethanol extract from Atractylodes macrocephala （EEAM） on microglial phagocytosis and degradation of amyloid β （Aβ） based on peroxisome proliferator-activated receptor γ （PPAR- γ） signaling pathway. METHODS Taking neuromicroglial cell BV2 as subjects， confocal microscopy was used to observe the effects of EEAM （0.3， 0.4， 0.5 mg/mL， similarly hereinafter） on phagocytosis and degradation of Aβ in microglia. Human embryonic kidney cell HEK293 was used to investigate the effects of EEAM on luciferase transcriptional activity of PPAR-γ. The effect of EEAM on nuclear translocation of PPAR-γ was investigated by immunofluorescence. Alzheimer’s disease BV2 cell model was induced by Aβ1-42， and quantitative polymerase chain reaction was used to investigate the effects of EEAM on mRNA expressions of PPAR-γ downstream target genes （Lxra， Lxrb， Abca1， Abcg1， Cd36， Sra and Apoe）. RESULTS The results of Aβ uptake experiment showed that after the intervention of medium and high doses of EEAM， fluorescence intensity of Aβ in BV2 cells increased significantly （P＜0.05）. The degradation experiment of Aβ showed that after the intervention of medium and high doses of EEAM， fluorescence intensity of Aβ in BV2 cells decreased significantly （P＜0.05）. After the intervention of different doses of EEAM， luciferase transcriptional activity of PPAR-γ in HEK293 cells increased significantly （P＜0.05）； fluorescence intensity of PPAR-γ in BV2 cells and nuclei （except for low-dose group） increased significantly （P＜0.05）. mRNA expressions of Lxra， Lxrb， Abca1， Abcg1， Cd36， Sra and Apoe in BV2 cells were increased significantly （P＜0.05）. CONCLUSIONS EEAM can promote the uptake and degradation of Aβ in microglia by activating PPAR-γ signaling pathway， thus improving Alzheimer’s disease.
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OBJECTIVE To explore the mechanism of Yishen tongluo formula （YSTLF） in improving abnormal lipid metabolism based on the sterol regulatory element binding proteins （SREBPs） pathway. METHODS Using C57BLKS/J （db/db） mice as model and C57BLKS/J （db/m） mice as normal control， the mechanism of 1， 2.5 and 5 g/kg YSTLF improving abnormal lipid metabolism of db/db mice was investigated by determining the liver coefficient， the contents of serum total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein （LDL） and high-density lipoprotein （HDL）， observing steatosis and lipid accumulation in liver tissue of mice， detecting the protein expressions of SREBP-1 and SREBP-2 as well as mRNA transcription levels of Srebp- 1c， Srebp-2 and their downstream lipid metabolism-related target genes （Fasn， Acc1， Scd5， Fads1， Hmgcr， Dhcr24， Insig-1， Fdps） in liver tissue of mice. Using low-fat cultured human liver cancer cell HepG2 as an in vitro cell model for abnormal lipid metabolism， and 25-HC （SREBPs inhibitor， 10 μmol/L） as the control， the effects of 125， 250 and 500 μg/mL YSTLF on protein expressions of SREBP-1 and SREBP-2 as well as mRNA transcription of SREBP-1c， SREBP-2 and their downstream lipid metabolism-related target genes were investigated to verify the mechanism in vitro. RESULTS 1， 2.5， 5 g/kg YSTLF significantly reduced the levels of TC， TG and LDL， the percentage of lipid droplet-positive region in liver tissue and liver coefficient， significantly down-regulated protein expressions of Pre-SREBP-1， n-SREBP-1， Pre-SREBP-2 and n-SREBP-2， and mRNA transcription of Srebp-1c， Srebp-2 and their downstream target genes in liver tissue， while significantly increased HDL level， with statistical significance （P＜0.05 or P＜0.01）. In the cell experiment in vitro， the expressions of the above-mentioned proteins and genes in the cells treated with YSTLF at 125， 250 and 500 μg/mL for 24 hours were consistent with those in the animal experiment； there was no significant difference in the expressions of the above-mentioned proteins and genes between inhibitor control group and 250， 500 μg/mL YSTLF groups （P＞0.05）. CONCLUSIONS YSTLF can regulate the expression of transcription factor SREBPs， so as to inhibit the high expression of fatty acid and cholesterol synthesis-related genes， promote the degradation of TC and TG， improve the abnormality of lipid metabolism and inhibit lipid accumulation， thus playing the role of lipid-lowering.
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OBJECTIVE To study the effects of ginsenoside Rb 1（G-Rb1）on epithelial-mesenchymal transition （EMT）of renal tubular epithelial cells and its potential mechanism. METHODS The growth factor β1（TGF-β1）10 ng/mL was used to induce EMT of human renal tubular epithelial cells HK- 2. The morphological changes of HK- 2 cells were observed after treated with 10， 20，30 μmol/L G-Rb1 for 48 h. The transcriptional activities of biovector SBE in human embryonic kidney cell HEK 293 were determined after 24 h treatment with 1.0，2.5，5.0，10，20，30 μmol/L G-Rb1. Effects of above concentration of G-Rb 1 on the viability of HK- 2 cells were determined after 24 h of treatment. mRNA expressions of α-smooth muscle actin （α-SMA），collagen Ⅰ （COL-Ⅰ）and fibronectin （FN）in HK- 2 cells were detected after treated with 10，20，30 μmol/L G-Rb1 for 24 h. The expressions of α-SMA，Smad3，p-Smad3，COL-Ⅰ，FN and E-cadherin were detected after treated with 10，20，30 μmol/L G-Rb1 for 24 h. RESULTS G-Rb1 of 10-30 μmol/L significantly inhibited TGF-β1-induced EMT in HK- 2 cells and the increase of transcriptional activities of biovector SBE induced by TGF-β1（P＜0.05），but had no effects on relative activities of HK- 2 cells（P＞0.05）. The protein and mRNA expressions of α-SMA，COL-Ⅰ and FN ， the protein expressions of Smad 3 and p-Smad 3 were significantly up-regulated induced by TGF-β1（P＜0.05），while the protein expression of E-cadherin was significantly down- regulated（P＜0.05）；G-Rb1 could effectively reverse aboveprotein or mRNA expressions. CONCLUSIONS G-Rb1 can protect renal tubular epithelial cells from EMT induced byxiezhishen TGF-β1 to a certain extent ，which may be related to inhibiting the activation of TGF-β1/Smad3 signaling pathway.
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Objective:To evaluate the risk factors for prognosis of primary intussusception treated by enema reduction in children.Methods:A retrospective analysis of 519 patients with primary intussusception in Anhui Provincial Children′s Hospital from January 2017 to December 2020 was performed. Among the 519 patients, there were 346 males and 173 females. The median age was 15 months, with a range of 3 to 69 months, some basic information was collected. Patients were divided into bad prognosis group ( n=71) and good prognosis group ( n=448) according to prognosis.Propensity score matching (PSM) was conducted to reduce confounding bias between the groups. Conditional logistic regression were used to analysis the risk factors of primary intussusception treated by enema reduction. Results:There were 71 cases of poor prognosis among 519 primary intussusception patients, incidence was 13.7%.Before PSM, there was significant difference in 6 covariates, including gender, age, duration of symptoms, bloody stool, diarrhoea and fever between two group ( P<0.05). There was no significant difference in abdominal pain and vomit between two group ( P>0.05). With propensity score matching, 69 pairs of patients were sucessfully matched. After PSM, distribution of the above covariates reached equilibrium between two groups ( P>0.05). There were statistically significant differences in enema reduction, mass location, seroperitoneum, lactic acid and overweight/obese between the two groups ( P<0.05). Conditional logistic regression analysis confirmed that enema reduction( OR=3.478, 95% CI: 1.150-10.517, P=0.027), mass location ( OR=6.596, 95% CI: 1.669-26.056, P=0.007), lactic acid( OR=1.012, 95% CI: 1.003-1.021, P=0.010), overweight/obese ( OR=6.085, 95% CI: 1.650-22.436, P=0.007) were independent factors for predicting prognosis of primary intussusception treated by enema reduction. Conclusions:AER, mass located left hemicolon, elevated lactic acid and overweight/obese were independent risk factors for poor prognosis of primary intussusception treated by enema reduction.
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Objective:To explore the clinical characteristic, pathogenesis, diagnosis and treatment strategies of gastroinstinal injury caused by magnets ingestion in children.Methods:A retrospective analysis of 46 patients with gastrointestinal tract magnets ingestion in Anhui Provincial Children′s Hospital from October 2017 to September 2021 was performed. Patients were divided into different groups according to gastroinstinal perforation. Some basic information was collected, including gender, age, duration of swallow foreign bodies, quantity of foreign bodies, symptoms, white blood cell, neutrophil, C-reactive protein, therapeutic method, gastroinstinal injury and follow up. Logistic regression. Univariate analysis and multivariate Logistic regression were used to analysis the risk factors of gastroinstinal perforation. Continuous parametric data were summarized using median and interquartile range, differences were evaluated using Wilcoxon Mann-Whitney test.Noncontinuous data were analyzed using chi-square test or corrected chi-square test.Results:Among the 46 patients, there were 33 males and 13 females. The median age was 3 years, with a range of 8 months to 11years. 34 cases had a history of ingesting magnets. The common number of ingested magnets was 2-10 (25 cases). Vomiting (18 cases) and abdominal pain (13 cases)were the commonest complaint. 6 cases tend to pass through the gastrointestinal tract uneventfully, and the remaining cases were successfully extracted by endoscopy (5 cases) and surgery(35 cases). Gastroinstinal perforation was found in 28 cases, and the majority of perforation was located in the ileum (18 cases). Univariate analysis showed that symptom, white blood cell, neutrophil, and CRP were associated with gastroinstinal perforation ( P<0.05). Multivariate Logistic regression analysis showed that symptom( OR=4.715, 95% CI: 1.074-20.696, P=0.040) and CRP( OR=11.605, 95% CI: 1.132-118.981, P=0.039) were independent factors for gastroinstinal perforation. There was no significant correlation between the number of magnets and gastroinstinal perforation ( r=0.276, P>0.05). Conclusions:The ingestion of magnetic foreign bodies in children often requires urgent management.When magnets are located within the prepyloric part of the GI tract, retrieval by endoscopy is recommended.The surgical intervention is required as soon as possible due to the failure of endoscopic treatment.
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ObjectiveTo investigate the protective effect of Liuwei Dihuangwan on neurovascular injury in SAMP8 mice. MethodThe Alzheimer's disease （AD） model with insufficiency of kidney essence was induced in 75 SAMP8 mice aging 6 months. The model mice were divided into model group， positive control group （donepezil hydrochloride， 0.747 mg·kg-1·d-1）， and high-， medium-， and low-dose Liuwei Dihuangwan groups （2.700， 1.350， 0.675 g·kg-1·d-1）， with 15 mice in each group. Fifteen SAMR1 mice were assigned to a normal control group. All mice were administered continuously for 2 months. The spatial memory of mice was tested by the Morris water maze. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in the hippocampus and cortex of brain tissues. The immunohistochemical method （IHC） was used to detect the deposition of amyloid β-protein （Aβ） and the expression of von Willebrand factor （vWF） and CD34 in the hippocampus and cortex of brain tissues. Electron microscopy was used to observe the ultrastructural changes in cerebral microvessels. Western blot was used to detect the protein expression levels of the receptor of advanced glycation endproduct （RAGE）， low-density lipoprotein receptor-related protein 1 （LRP1）， vascular endothelial growth factor A （VEGF-A）， and P-selection in the hippocampus and cortex of brain tissues. ResultCompared with the normal control group， the model group showed prolonged escape latency and swimming distance （P<0.01）， increased number of glial cells， decreased number of nerve cells， blurred tight junctions or enlarged gap of the brain microvascular endothelial cells， severely injured membrane structure， swollen mitochondria of endothelial cells， ruptured membrane， massive dissolution in cristae， increased protein expression of Aβ and vWF in the hippocampus and cortex （P<0.01）， reduced protein expression of CD34 （P<0.05）， elevated protein expression of RAGE and P-selection in the cortex （P<0.01）， and decreased protein expression level of LRP1 and VEGF-A （P<0.01）. Compared with the model group， the Liuwei Dihuangwan groups showed shortened escape latency and swimming distance （P<0.05）， reduced number of glial cells in the cortex and hippocampus， increased number of microvessels in the cortex， clear double-layer membrane structure in tight junctions between the microvascular endothelial cells， increased number of mitochondria with intact membrane and recovered mitochondrial cristae， decreased protein expression of Aβ， vWF， RAGE， and P-selection in the hippocampus and cortex （P<0.05）， and increased protein expression of CD34， LRP1， and VEGF-A （P<0.05）. ConclusionLiuwei Dihuangwan can regulate Aβ metabolism through the RAGE/LRP1 receptor system and promote cerebral microvascular angiogenesis by inhibiting vWF expression and increasing VEGF-A and CD34， thereby improving cerebral microvascular injury in SAMP8 mice.
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ObjectiveTo investigate the effect of Liuwei Dihuangwan on memory function of senescence-accelerated mouse prone 8 （SAMP8） mice by regulating autophagy through the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/forkhead box O3a （FoxO3a） pathway. MethodSix male senescence-accelerated mouse resistant 1 （SAMR1） mice of SPF grade aging 6 months were assigned to a normal group， and 24 male SAMP8 mice of SPF grade aging 6 months were randomly divided into a model group， a donepezil group （0.747 mg·kg-1）， and high- and low-dose Liuwei Dihuangwan groups （2.700 and 1.350 g·kg-1）， with 6 mice in each group. The mice were treated with drugs by gavage for 2 months. Morris water maze was used to detect the learning and memory abilities of mice in each group. Nissl staining was used to observe the neurons in the cortex and hippocampus. The positive expression of microtubule-associated protein 1 light chain 3B （LC3B） in the cortex and hippocampus was detected by immunohistochemistry （IHC）. Western blot was used to detect the protein expression of the mammalian ortholog of yeast ATG6 （Beclin-1）， B cell lymphoma-2 （Bcl-2）， autophagy-related gene 5 （ATG5）， cysteinyl aspartate-specific protease 3 （Caspase-3）， Caspase-9， Akt， p-Akt， FoxO3a， and p-FoxO3a. ResultCompared with the normal group， the model group showed prolonged escape latency （P<0.05，P<0.01）， reduced number of platform crossings and the residence time in the target quadrant （P<0.01）， decreased neurons with reduced volume and dispersed distribution in the cortex and hippocampus， increased positive expression of LC3B （P<0.01）， elevated expression of Beclin-1 and ATG5 in the cortex （P<0.01）， declined Bcl-2 expression （P<0.01）， up-regulated Caspase-3 and Caspase-9 expression （P<0.01）， and decreased expression levels of p-Akt/Akt and p-FoxO3a/FoxO3a （P<0.01）. Compared with the model group， the donepezil group and the Liuwei Dihuangwan groups showed shortened 3 d escape latency （P<0.05，P<0.01）， increased number of platform crossings （P<0.01）， and prolonged residence time in the target quadrant （P<0.01）. In the donepezil group， the number of neurons in the cortex and hippocampus was increased. In the Liuwei Dihuangwan groups， the number of neurons and Nissl bodies increased with denser distribution and lower degree of cell damage. The positive expression of LC3B in the cortex and hippocampus was decreased in the donepezil group and Liuwei Dihuangwan groups （P<0.01）. The expression of Beclin-1 was decreased in the Liuwei Dihuangwan groups （P<0.01）. The expression of ATG5 was decreased in the donepezil group and the low-dose Liuwei Dihuangwan group （P<0.01）. The donepezil group and the Liuwei Dihuangwan groups showed the increased expression level of Bcl-2 in the cortex （P<0.01）， decreased expression level of Caspase-3 （P<0.01）， reduced expression level of Caspase-9 （P<0.05，P<0.01）， and elevated expression levels of p-Akt/Akt and p-FoxO3a/FoxO3a （P<0.01）. ConclusionLiuwei Dihuangwan can effectively improve the learning and memory abilities of the SAMP8 mice and protect neurons. Its mechanism may be related to the regulation of the PI3K/Akt/FoxO3a signaling pathway， down-regulation of the expression of ATG5， Beclin-1， and LC3B， and the inhibition of apoptosis.
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OBJECTIVE： To study the effects of Atractylodes macrocephala ethanol extract （AM） on life span of Caenorhabditis elegans（called N 2 nematode for short ），and to investigate its mechanism based on transcription factor SKN- 1/ nuclear factor E 2 related factor 2（Nrf2）. METHODS ：N2 nematode were divided into blank control group ，positive control group （100 μ mol/L curcumin，similarly hereinafter ），AM low-dose ，medium-dose and high-dose groups （100，200，300 μ g/mL， similarly hereinafter ）. The effects of AM on the life span （by average survival time ）of N 2 nematode under normal condition and oxidant stress condition （40 mmol/L H 2O2）as well as its effects on reproductive capability （by the number of filial generation ）of N2 nematode under normal condition were investigated . 700 μmol/L H2O2 was used to establish neuroblastoma cells N 2a oxidant stress model. Effects of positive control ，low-dose，medium-dose and high-dose of AM on the survival rate of model cells were detected by MTT method. After human embryonic renalepithelial cells 293T were transfected with Nrf 2-ARE plasmid ， the effects of positive control and AM on luciferase activity of Nrf2-ARE were detected by luciferase reporter gene method at low，medium and high dose for 24 h and at medium dose for 12，18 and 24 h. RT-PCR was used to detect the effects ofpositive control ，low-dose，medium-dose and high-dose of AM on the mRNA expression of downstream genes NQO- 1 and HO- 1 of Nrf 2 in N 2a cells as well as mRN A expression of en@hactcm.edu.cn downstream genes GCS- 1，GST-7，GST-10，HSP-60，HSP- 16.2 and SOD- 3 of SKN- 1 in N 2 nematode. RESULTS ：Compared with blank control group ，average survival time of N 2 nematode under normal and oxidant stress condition was significantly prolonged in positive control group and AM groups ；the number of filial generation on the first day （except for AM high-dose group ），the number of filial generation on the second day （except for AM low-dose group ） and the total number of filial generation （except for AM low-dose group ） were increased significantly（P＜0.05 or P＜0.01）. The survival rate of N 2a cells in positive control group ，AM medium-dose and high-dose groups were significantly higher than that of model group （P＜0.05 or P＜0.01）. Compared with blank control group ，Nrf2-ARE luciferase relative activity of 293T cells in positive control group and AM groups as well as Nrf 2-ARE luciferase relative activity of 293T cells in AM medium-dose group after different time of treatment were increased significantly （P＜0.01），in dose-dependent and time-dependent trend. Compared with blank control group ，mRNA relative expression of HO- 1 and NQO- 1（except for positive control group ），GCS-1（except for AM low-dose group ），GST-7（except for positive control group and AM low-dose group ）， GST-10 and HSP- 60（except for AM low-dose group ），HSP-16.2（except for positive control group and AM low-dose group ）and SOD-3 （except for positive control group and AM low-dose group ） were increased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS：AM can prolong the life span of N 2 nematode under normal and oxidant stress condition and improve the its reproductive capacity ，the mechanism of which may be associated with the activation of SKN- 1/Nrf2 signaling pathway.
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Objective To evaluate the diagnostic value of magnetic resonance enterography(MRE)and ectopic gastric mucosa imaging(EGMI)in children with lower gastrointestinal hemorrhage.Methods The clinical data including gender,age,amount of bleeding and preoperative minimum hemoglobin(Hb)levels of 54 children with lower gastrointestinal hemorrhage were collected,who received surgical exploration in Department of Pediatric Surgery,Anhui Provincial Children's Hospital between February 2014 and April 2016.Children were divided into lesion group and non-lesion group according to the findings of surgical exploration.Lesion group were defined as children with Meckel's diverticulum(MD)or duplication of the small intestine by surgery and postoperative pathological findings.A total of 36 cases,25 cases of boys,11 cases of girls,aged(2.86±1.59)years old.Non-lesion group were defined as children,who underwent operation with negative result or with lesions but not MD or duplication of the small intestine.A total of 18 cases,14 cases of boys,4 cases of girls,aged(3.87±1.62)years old.MRE and EGMI were performed when the children stopped bleeding,before the inspection,should fasting for 4-6 h.SPSS 13.0 software was used for statistical processing.The evaluation of EGMI,MRE and the both in diagnosing MD or duplication of the small intestine were conducted by receiver operating characteristic curve(ROC).According to the principles of biggest gain best diagnostic value by Youden index,and the degree of sensitivity and specificity was calculated at this time,P0.05).There were statistically significant differences in age,amount of bleeding and preoperative minimum hemoglobin levels(t=2.179,2.027,2.222,all P<0.05).There were statistically significant differences in classification comparison by EGMI and MRE between 2 groups(x2=15.226,29.121,all P<0.01).The optimal cut-off value of EGMI and MRE in the diagnosis of enteric malformation was more than level 3,and the suspected positive and being positive value was assigned as positive results.According to the cut-off value above,EGMI,MRE and EGMI plus MRE in series and in parallel in the diagnosis of enteric malformation,the areas under curves(AUC)were 0.809,0.917,0.750 and 0.847,respectively.The sensitivity was 61.1％,75.0％,55.6％and 80.6％,respectively.The specificity was 90.4％,94.4％,94.4％and 88.9％,respectively.The Youden's indexes were 0.515,0.694,0.500 and 0.695,respectively.The optimal cut-off value of age and preoperative minimum hemoglobin levels in the diagnosis of enteric malformation were 2.92 years and 80 g/L,respectively.The AUC was 0.761 and 0.672.The Youden's indexes were 0.515 and 0.333.There was no significant differences statistically in AUC compared with age,MRE and EGMI(all P<0.05).Conclusions The high diagnostic value in children with lower gastrointestinal hemorrhage is found by using MRE and EGMI.Then,MRE as a separate diagnosis method is also worthy of clinical application.
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Objective:To prepare and identify the mouse anti-human monoclonal antibodies ( mAbs) using leukocytes as im-munogens. Methods: The mice were immunized using human peripheral blood leukocytes. Then, use of B lymphocyte hybridoma technology preparation of mAbs,followed screening by immunocytochemistry and limited dilution. The secreted mAbs were identified by immunoprecipitation,mass spectrometry,Western blot,ELISA and immunohistochemistry. Results:The 35 positive polyclonal cells were obtained,of which 11 strains secreted mAbs against S100A9. And one strain was used to prepare monoclonal antibody. The purified mAb against S100A9 were purified and identified as IgG1 subtype,with the titer,purity and affinity constant was 1∶3. 18×105,95% and 3. 54×108 L/mol,respectively. This mAb generally had 0. 12% crossed reactivity to S100A8 ,and showed little or no cross reactivity to S100A12 and S100A13. The prepared monoclonal antibodies can specifically recognizes the S100A9 antigen in human breast cancer tissues. Conclusion:Successful preparation of mAb against S100A9,which can secrete specific mAb against S100A9 protein with high titers and specificity have been established successfully,which laid the foundation for the immunology application.
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BACKGROUND:When acute cerebral ischemia attacks, matrix metaloproteinases (MMPs) lead to the occurrence of cerebral edema through degrading the extracelular matrix, breaking the close connection between endothelial cels, increasing the permeability of capilaries, and destroying the blood brain barrier. OBJECTIVE: From the aspects of MMPs and extracelular matrix, to discuss the therapeutic effects ofbuyang huanwu decoction combined with bone marrow mesenchymal stem cel (BMSCs) transplantation on cerebral ischemia-reperfusion injury. METHODS:A total of 96 Sprague-Dawley rats were randomly divided into model group, tissue inhibitor of MMPs-1 (TIMP-1) group, TIMP-1+BMSCs group (BMSCs group) andbuyang huanwu decoction+BMSCs+TIMP-1 group (combined group that was divided into four subgroups, 12-, 24-, 36-, 48-hour groups). Rat models of cerebral ischemia-reperfusion were constructed, and TIMP-1 and BMSCs were injected to the brain of rats by a microinjector in a stereotaxic apparatus. Rats in the combined group were given buyang huanwu decoction (10 mL/kg), and rats in the other groups were given the same volume of normal saline at 7 days before surgery. After 10 days of administration, serum samples and brain tissues were colected to determine MMP-2 and MMP-9 levels using ELISA and to detect MMP-9 activity using gelatinases spectrometry method. RESULTS AND CONCLUSION:Compared with the model group, the levels of MMP-2 and MMP-9 in the serum and MMP-9 activity in the brain were decreased in the other groups to different extents, especialy the levels of MMP-9 (P < 0.05). Compared with the BMSCs group, the levels of MMP-2 and MMP-9 in serum as wel as activities of MMP-9 and pro-MMP-9 in the brain were decreased significantly in the combined group at 36 and 48 hours after treatment (P< 0.01). The results show that thebuyang huanwu decoction can be mutualy cooperated with TIMP-1 to inhibit the degradation of extracelular matrix induced by MMP-2 and MMP- 9, repair the damaged blood brain barrier, prevent and cure cerebral edema after ischemia.
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BACKGROUND:Cerebral ischemia often occurs in underlying pathological conditions, such as hypertension, hyperlipidemia and diabetes. Therefore, it is of great significance to construct a cerebral ischemia rat model with hyperlipidemia and to study the effect of basic pathological changes on the cerebral ischemia. OBJECTIVE:To observe the brain tissue pathological changes of rat models with coexistence of hyperlipidemia and cerebral ischemia, and the effect of hyperlipidmia on cerebral ischemia. METHODS:The rats were fed with high-fat diet to establish the hyperlipidmia models, and then focal cerebral ischemia models were prepared with suture method. At 3 and 7 days after modeling, the 2,3,5-triphenyltetrazolium chloride staining was used to observe the volume of brain tissue ischemia, and hematoxylin-eosin staining was performed to observe pathological change of the margin of the brain tissue ischemia zone. RESULTS AND CONCLUSION:2,3,5-Triphenyltetrazolium chloride staining staining results showed that the volume of cerebral ischemia was significantly reduced in the hyperlipidemia+cerebral ischemia 7 day group. Hematoxylin-eosin staining results showed there was typical ischemic changes in al the cerebral ischemia models, and the number of microglial cel s after cerebral ischemia for 7 days was significantly smal er than that after cerebral ischemia for 3 days, and the changes were more obvious in the hyperlipidemia+7-day cerebral ischemia group when compared with the hyperlipidemia+3-day cerebral ischemia group. Ultrastructure showed there were neuronal and glial nuclear membrane shrinkage in al the cerebral ischemia models, mitochondria cristae was disappeared completely, endothelial cel mitochondria was decreased, most of the synaptic vesicles of nerve synapse were dissolved;the damages above were improved after ischemia for 7 days, especial y hyperlipidemia+cerebral ischemia for 7 days, the neuronal degeneration and necrosis were reduced, the mitochondrial damage was repaired, the number of mitochondrial cristae was increased significantly, and the synaptic vesicles of nerve synapse were recovered significantly. The results indicate that hyperlipidemia can promote the recovery of cerebral ischemic injury, probably because the hyperlipidemia factors can activate the protection mechanism.
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BACKGROUND: It is demonstrated in modern pharmacologic study that total paeony glycoside (TPG) provides extensive pharmacologic activities,such as inhibiting aggregation of platelets and erythrocytes, anticoagulation,antithrombsis, anti-arterial sclerosis, protecting heart and liver, anti-tumor,etc.OBJECTIVE: Neonatal rat cardiomyocytes were cultured in vitro and by the changes of superoxide dismutase (SOD) activity, malondialdehyde (MDA) and nitric oxide (NO) contents in cell culture solution, the protection of TPG on injured cardiomyocytes was analyzed.DESIGN: Controlled observation was designed.SETTING: Bioengineering Department in School of Life Science and Technology of Xi 'an Jiaotong University and Institute of Bone Diseases in Medical School of Xi'an Jiaotong University.MATERIALS: The experiment was performed in Bioengineering Department in School of Life Science and Technology of Xi 'an Jiaotong University from February to June in 2003, in which, 44 SD neonatal rats aged 1-3 days were employed. The 48-hour-cultured cardioryocytes were prepared in 42 bottles and randomized into 6 groups, named normal control (normal group), medicated-injury group (injury group), TPG 0.625 mg/L group,TPG 3.125 mg/L group, TPG 15.625 mg/L group and positive control, 7 bottles in each group.METHODS: Cardiomyocytic primary culture was performed under aseptic condition. No any drug was used in normal group, isoprenaline was added in injury group to terminate the concentration at 100 mg/L, in TPG 0.625, 3.125 and 15.625 mg/L groups, 30 minutes after isoprenaline added, RGP at dosages of 0.625, 3.125 and 15.625 mg/L were added respectively; in positive control, 30 minutes after isoprenaline added, coenzyme Q10 was used to terminate the concentration as 100 mg/L.Afterwards, the assay of every index was performed. Xanthine oxidase (XOD) method was used to assay SOD activity, thiobarbituric acid (TBA)method was to assay MDA content and nitrate reductase (NR) method was to assay NO content.cell culture solution in each group.Compared with normal group, the levels of total SOD, CuZn-SOD and MnSOD were reduced remarkably in injury group (P ＜ 0.05 or P ＜ 0.01).The above-indexes in every TPG group and positive control were improved to different extents (P ＜ 0.05 or ＜ 0.01), in which, the protection of TPG 15.625 mg/L group was near to or superior to positive control [(79.50±10.67), (80.30±13.50); (48.24±13.26), (49.73±10.23); (31.26±10.22),in cell culture solution in each group: Those in injury group were higher remarkably than normal group (P ＜ 0.01). MDA and NO contents were all reduced in every TPG group and positive control and dose dependence presented in TPG protection, the higher the dose was, the stronger the action of TPG on protection was, in which, in high-dose group, MDA content was near to normal group [(5.41±1.81), (4.48±0.94) μmol/L, P ＞ 0.05] and NO content was similar to positive control [(81.83± 9.08), (82.41±12.37) mol/L,P ＞ 0.05].CONCLUSION: TPG protects myocardial injury induced by isoprenaline,indicating dose-dependence relationship, which is probably associated with enhanced anti-oxidation of cell, reduced injury of cellular membrane induced by free radical and lipid oxidant.
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Aim To investigate the antioxidative effect of total paeony glycoside(TPG) on cardiomyocytes injured.Methods The ischemia and hypoxia injuy model of cultured neonatal rat cardiomyocytes was induced by adding isoprenaline(ISO), and superoxide dismutase(SOD), malonalde hyde(MDA) and nitric oxide(NO) in the culture solution of normal control group; ISO injure group, CoQ 10 positive control group as well as protective group s with high,middle or low-dose TPG were respectively analyzed and compared. Results Compared with normal control group,the enzyme activity o f total SOD, CuZn-SOD and Mn-SOD decreased obviously, and the content of MDA and NO increased markedly in injury group, but in TPG and CoQ 10 groups all of detective indicators had improvement in varying degrees, and the protective effect was better than or close to positive control group in high-dose TPG grou p.Conclusion TPG has protective action on injured cardiomyocyte s induced by ISO in dose-dependent manner. The mechanism relates to the enhance ment of antioxidative effect in cells, and the reduction of membrane damage caus ed by free radical and lipid peroxide.
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Urine glycosaminoglycans of two patients (full brother and sister )with Sanfilippo's syndrome were analysed by electrophoresis, enzymatic or chemical treatment and ion-exchange chromatography. Heparin sulfate is the main glycosaminoglycan accouting for 70% and 58% of urine glycosaminoglycans in both patients, whereas only 29% and 42% of urine glycosaminoglycans are cnondroitin sulfate. Tbe C/O ratio urine heparin sulfate of the patients is 2.32, indicating that the hexuronic acid component parts of the patient's heparin sulfate are much the same as general heparin sulfate. However, the analysis of glycosaminoglycans with DEAE-Sephadex A-25 chromatography and electrophoresis in 0.1 M HCL showed that urine heparin sulfate of the patients arc heterogeneous, and that it covers, at least, two molecular types with different amount of sulfate and charge density.