Effect and mechanism of hyperthermia on cell apoptosis of laryngeal squamous cell carcinoma patients / 中华放射肿瘤学杂志

Objective:To evaluate the effect of hyperthermia on the apoptosis and the expression levels of cysteine-containing aspartate-specific protease-3 (Caspase-3) and phosphorylated protein kinase B (p-AKT) in laryngeal squamous cell carcinoma cells.Methods:A prospective study was conducted on 30 patients with laryngeal squamous cell carcinoma who were treated at the First Affiliated Hospital of Zhengzhou University from October, 2021 to October, 2022. Three times of hyperthermia were performed with a time interval of 24 h. The tumor tissue samples were collected from 30 patients before and after hyperthermia and divided into before hyperthermia group (group A ) and after hyperthermia group (group B). Self-control study mode was adopted for comrparative analysis. The cell apoptosis was detected by TUNEL assay. The expression levels of Caspase-3 and p-AKT in the tissues were detected by immunohistochemistry. Positive cell ratio and immunohistochemistry (IHC) score were recorded. Comparison between two groups was performed by paired t-test. The correlation between the degree of apoptosis and the changes of Caspase-3 and p-AKT molecules was assessed by Pearson correlation analysis. Results:No evident adverse reactions were observed in 30 patients after hyperthermia. The apoptosis index of laryngeal squamous cell carcinoma cells in group A was 2.37%±1.33%, and 4.27%±3.93% in group B ( P=0.006). In group A, the ratio of Caspase-3 positive cells in tumor tissues was 62.31%±19.49% and 80.79%±17.15% in group B ( P=0.001). The ratio of p-AKT positive cells in group A was 31.26%±19.30%, and 26.26%±15.86% in group B ( P=0.023). There was a positive correlation between the degree of apoptosis and the changes of Caspase-3 molecule ( r=0.544, P=0.002), but a negative correlation was noted between the degree of apoptosis and the changes of p-AKT molecule ( r=-0.434, P=0.017). Conclusion:Hyperthermia can promote the apoptosis of tumor cells in laryngeal squamous cell carcinoma, which may be related to Caspase-3 dependent apoptosis, and the inhibition of AKT phosphorylation is also involved in this process.

Jiedu Huoxue Prescription Regulates NF-κB/NLRP3/Caspase-1 to Promote Vascular Reendothelialization in Rat Model of Injured Thoracic Aorta / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Jiedu Huoxue prescription in promoting the reendothelialization of injured vessels by regulating the nuclear factor （NF）-κB/NOD-like receptor protein 3 （NLRP3）/cysteine-aspartic acid protease （Caspase）-1-mediated pyroptosis. MethodA rat model of injured thoracic aorta was established by balloon injury， and 36 rats were assigned into shame surgery， model， low-， medium-， and high-dose Jiedu Huoxue prescription， and atorvastatin calcium tablet groups. The injured aortic segment was collected 28 days after surgery. Hematoxylin-eosin （HE） staining and Evans blue staining were conducted to reveal the changes of vascular structural morphology and the reendothelialization of blood vessels， respectively. The enzyme-linked immunosorbent assay （ELISA） was employed to determine the levels of tumor necrosis factor-α （TNF-α）， intercellular adhesion molecule-1 （ICAM-1）， interleukin （IL）-1β， and nitric oxide （NO） in the serum. Western blotting was employed to determine the expression of endothelial nitric oxide synthase （eNOS）， NF-κB p65， phospho-NF-κB p65 （p-NF-κB p65）， NLRP3， and Caspase-1 in the vascular tissue. ResultThe model group showed thickened endovascular membrane， proliferation and disarrangement of smooth muscle cells of the artery wall， obvious inflammatory cell infiltration， and narrowed luminal area. Jiedu Huoxue prescription and atorvastatin calcium tablets mitigated the pathological changes of the thoracic aorta in different degrees. After balloon injury， the endothelial coverage rate of the model group decreased significantly， while Jiedu Huoxue prescription and atorvastatin calcium tablets increased the reendothelialization rate （P<0.05）. Compared with the shame surgery group， the model group showed elevated levels of TNF-α， ICAM-1， and IL-1β （P<0.01） and lowered NO level （P<0.01） in the serum. In addition， the model group presented down-regulated protein level of eNOS （P<0.01） and up-regulated phosphorylation of pyroptosis-associated proteins NLPR3， Caspase-1， and NF-κB p65 in the vascular tissue （P<0.05， P<0.01）. Compared with the model group， Jiedu Huoxue prescription and atorvastatin calcium tablets lowered TNF-α， ICAM-1， and IL-1β levels （P<0.05， P<0.01） and elevated the NO level in the serum （P<0.05， P<0.01）. Moreover， the drugs up-regulated the expression of eNOS （P<0.01） and down-regulated the expression of NLRP3， Caspase-1， and NF-κB p65 （P<0.05， P<0.01） in the vascular tissue. ConclusionJiedu Huoxue prescription can promote the reendothelialization and inhibit the intimal hyperplasia of vessels after balloon injury by regulating the NF-κB/NLRP3/Caspase-1 pathway to inhibit pyroptosis and reduce endothelial inflammatory injury.

Preliminary study on 99Tc m-3PRGD 2 imaging to verify the anti-angiogenesis mechanism and efficacy of Mongolian medicine Sendeng-4 decoction for rheumatoid arthritis / 中华核医学与分子影像杂志

Objective:To explore the therapeutic mechanism of Mongolian medicine Sendeng-4 decoction for rheumatoid arthritis by 99Tc m-hydrazinonicotinamide-(polyethylene glycol) 4-E[(polyethylene glycol) 4-c((Arg-Gly-Asp)fk)] 2 (3PRGD 2) imaging. Methods:A total of 200 female SD rats (age: 6-7 weeks) were divided into collagen-induced arthritis (CIA) group ( n=176) and blank control group ( n=24). Rats in the CIA group were divided into Sendeng-4 decoction treatment group ( n=24), etanercept treatment group ( n=24), and negative control group ( n=24) by simple random sampling method. 99Tc m-3PRGD 2 SPECT/CT imaging was performed before and after modeling and treatment. The differences of target/non-target (T/NT) ratio and serological, pathological, and immunohistochemical results among groups were compared by one-way analysis of variance or Kruskal-Wallis rank sum test. The correlation was analyzed by Pearson correlation or Spearman correlation analysis. Results:There were 95 (95/176) CIA models successfully established. The T/NT ratios of Sendeng-4 decoction treatment group and etanercept treatment group were lower than that of negative control group (0.260± 0.094, 0.238±0.099, 0.766±0.144 ; F=163.00, P<0.001), while there was no significant difference between the two drug treatment groups ( P>0.05). After drug treatment, serum levels of vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α) and α vβ 3 were significantly lower than those of negative control group ( F values: 49.43-92.36, all P<0.001), pathological score was also lower than that of negative control group ( H=34.25, P<0.001), and levels of immunohistochemical makers (VEGF, TNF-α, α vβ 3, CD31, CD34) were also lower than those of negative control group ( H values: 13.51-26.84, all P<0.001), while there were no significant differences between the two drug treatment groups (all P>0.05). The T/NT ratios were positively correlated with above indictors in Sendeng-4 decoction treatment group ( r values: 0.56-0.59, rs values: 0.49-0.69), etanercept treatment group ( r values: 0.50-0.55, rs values: 0.46-0.70) and negative control group ( r values: 0.55-0.80, rs values: 0.58-0.86, P<0.001 or P<0.05). Conclusion:Verified by 99Tc m-3PRGD 2 SPECT/CT imaging and molecular pathology, Mongolian medicine Sendeng-4 decoction can inhibit neovascularization by down-regulating vascular factors such as VEGF, resulting in delaying the progression of the disease and improving clinical symptoms.

Prediction of pathological complete response to neoadjuvant chemotherapy in breast cancer using 99Tc m-3PRGD 2 imaging in comparison to 18F-FDG imaging / 中华核医学与分子影像杂志

Objective:To evaluate the value of 99Tc m-hydrazinonicotinamide-(poly-(ethylene glycol)) 4-E((poly-(ethylene glycol)) 4-c((Arg-Gly-Asp)fK)) 2 (3PRGD 2) imaging on predicting pathological complete response (pCR) outcomes to neoadjuvant chemotherapy (NAC) in patients with breast cancer and to compare it with 18F-FDG imaging. Methods:From October 2017 to October 2019, 41 patients (age: (61.5±7.8) years) who were diagnosed with stage Ⅱ and Ⅲ breast cancer and planned to receive preoperative NAC in the First Affiliated Hospital of Fujian Medical University and Xiehe Affiliated Hospital of Fujian Medical University were prospectively enrolled. All patients underwent both 99Tc m-3PRGD 2 and 18F-FDG imaging before NAC (baseline), and after the first and the fourth NAC cycle. The tumor/background ratio (T/B; 99Tc m-3PRGD 2) and SUV max ( 18F-FDG) in breast tumors and axillary lymph node (ALN) metastases were separately calculated. The relative T/B changes (ΔT/B 1, ΔT/B 2) and SUV max changes (ΔSUV max1, ΔSUV max2) after the first and the fourth NAC cycle compared to baseline were obtained. Patients underwent surgery after NAC and the pathology was used as the gold standard to determine whether patient achieved pCR. The predictive performance of ΔT/B and ΔSUV max regarding the identification of pCR or non-pCR was evaluated by using ROC analysis and the AUCs were compared by Delong test. Results:Of 41 patients, 13 (31.7%) were achieved pCR after NAC. For breast tumors, the AUCs of ΔT/B 1, ΔT/B 2, ΔSUV max1 and ΔSUV max2 were 0.827 ( P=0.001), 0.687 ( P=0.057), 0.859 ( P<0.001) and 0.713 ( P=0.030) respectively, and the AUCs of ΔT/B 1 and ΔSUV max1 had no significant difference ( z=0.33, P=0.740). For ALN metastases, the AUCs of ΔT/B 1, ΔT/B 2, ΔSUV max1 and ΔSUV max2 were 0.859 ( P=0.002), 0.778 ( P=0.014), 0.572 ( P=0.523) and 0.802 ( P=0.007) respectively, and the AUC of ΔT/B 1 was significantly higher than that of ΔSUV max1 ( z=2.10, P=0.035). Conclusion:The early relative changes of breast tumors and ALN metastases in 99Tc m-3PRGD 2 imaging during NAC can offer predictive information for pCR to NAC in patients with breast cancer, and early relative changes of ALN metastases in 99Tc m-3PRGD 2 imaging may have a higher predictive value for pCR than 18F-FDG imaging.

Prostate-derived IL-1β upregulates expression of NMDA receptor in the paraventricular nucleus and shortens ejaculation latency in rats with experimental autoimmune prostatitis / 亚洲男科学杂志(英文版)

Experimental autoimmune prostatitis (EAP)-induced persistent inflammatory immune response can significantly upregulate the expression of N-methyl-D-aspartic acid (NMDA) receptors in the paraventricular nucleus (PVN). However, the mechanism has not yet been elucidated. Herein, we screened out the target prostate-derived inflammation cytokines (PDICs) by comparing the inflammatory cytokine levels in peripheral blood and cerebrospinal fluid (CSF) between EAP rats and their controls. After identifying the target PDIC, qualified males in initial copulatory behavior testing (CBT) were subjected to implanting tubes onto bilateral PVN. Next, they were randomly divided into four subgroups (EAP-1, EAP-2, Control-1, and Control-2). After 1-week recovery, EAP-1 rats were microinjected with the target PDIC inhibitor, Control-1 rats were microinjected with the target PDIC, while the EAP-2 and Control-2 subgroups were only treated with the same amount of artificial CSF (aCSF). Results showed that only interleukin-1β(IL-1β) had significantly increased mRNA-expression in the prostate of EAP rats compared to the controls (P < 0.001) and significantly higher protein concentrations in both the serum (P = 0.001) and CSF (P < 0.001) of the EAP groups compared to the Control groups. Therefore, IL-1β was identified as the target PDIC which crosses the blood-brain barrier, thereby influencing the central nervous system. Moreover, the EAP-1 subgroup displayed a gradually prolonged ejaculation latency (EL) in the last three CBTs (all P < 0.01) and a significantly lower expression of NMDA NR1 subunit in the PVN (P = 0.043) compared to the respective control groups after a 10-day central administration of IL-1β inhibitors. However, the Control-1 subgroup showed a gradually shortened EL (P < 0.01) and a significantly higher NR1 expression (P = 0.004) after homochronous IL-1β administration. Therefore, we identified IL-1β as the primary PDIC which shortens EL in EAP rats. However, further studies should be conducted to elucidate the specific molecular mechanisms through which IL-1β upregulates NMDA expression.

Effects of ring finger and tryptophan-aspartic acid 2 on dendritic spines and synapse formation in cerebral cortex neurons of mice / 南方医科大学学报

OBJECTIVE@#To clarify the functional effects of differential expression of ring finger and tryptophan-aspartic acid 2 (RFWD2) on dendritic development and formation of dendritic spines in cerebral cortex neurons of mice.@*METHODS@#Immunofluorescent staining was used to identify the location and global expression profile of RFWD2 in mouse brain and determine the co-localization of RFWD2 with the synaptic proteins in the cortical neurons. We also examined the effects of RFWD2 over-expression (RFWD2-Myc) and RFWD2 knockdown (RFWD2-shRNA) on dendritic development, dendritic spine formation and synaptic function in cultured cortical neurons.@*RESULTS@#RFWD2 is highly expressed in the cerebral cortex and hippocampus of mice, and its expression level was positively correlated with the development of cerebral cortex neurons and dendrites. RFWD2 expression was detected on the presynaptic membrane and postsynaptic membrane of the neurons, and its expression levels were positively correlated with the length, number of branches and complexity of the dendrites. In cultured cortical neurons, RFWD2 overexpression significantly lowered the expressions of the synaptic proteins synaptophysin (P < 0.01) and postsynapic density protein 95 (P < 0.01), while RFWD2 knockdown significantly increased their expressions (both P < 0.05). Compared with the control and RFWD2-overexpressing cells, the neurons with RFWD2 knockdown showed significantly reduced number of dendritic spines (both P < 0.05).@*CONCLUSION@#RFWD2 can regulate the expression of the synaptic proteins, the development of the dendrites, the formation of the dendritic spines and synaptic function in mouse cerebral cortex neurons through ubiquitination of Pea3 family members and c-Jun, which may serve as potential treatment targets for neurological diseases.

Total Flavonoids of Peony Flower Regulate NLRP3 Inflammasome and Expression of Inflammatory Cytokines in Gouty Nephropathy Rats / 中国实验方剂学杂志

ObjectiveTo explore the possible mechanism of total flavonoids of peony flower （TFPF） in protecting rats from gouty nephropathy and provide data support for the pharmaceutical research on the treatment of gouty nephropathy. MethodGouty nephropathy rat model was established by adenine combined with ethambutol. Rats were randomly assigned into blank control group， model group， allopurinol （42 mg·kg-1） group， Tongfengshu tablets （600 mg·kg-1， positive control） group， and TFPF （260， 130， and 65 mg·kg-1） groups. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of tumor necrosis factor-α （TNF-α）， monocyte chemoattractant protein-1 （MCP-1）， interleukin-1β （IL-1β）， and interleukin-18 （IL-18） in rat serum and those of transforming growth factor-β1 （TGF-β1） and IL-1β in renal homogenate. Hematoxylin-eosin（HE） staining was carried out for observation of the morphological changes of renal cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） was conducted for observation of the DNA damage in renal cells. The expression of NOD-like receptor protein 3 （NLRP3）， cysteine aspartic acid protease（Caspase）-1 and IL-1β were observed by immunohistochemistry. The expression levels of NLRP3， Caspase-1 and nuclear transcription factor -κB （NF-κB） in renal tissues were detected by Western blot. ResultCompared with blank group， the contents of TNF-α， MCP-1， IL-1β， IL-18， and TGF-β1 in serum of model group were significantly increased （P<0.01）， and the expressions of NLRP3， Caspase-1， NF-κB and IL-1β in kidney of model group were significantly increased （P<0.01）. The renal tissue cells showed cytoplasmic swelling， cell membrane rupture， and the number of nuclear pyknotic fracture increased. The positive rate of TUNEL staining was significantly increased in model group （P<0.01）， and the contents of IL-1β and TGF-β1 in renal tissue homogenate were significantly increased （P<0.01）. Compared with model group， the contents of inflammatory factors TNF-α， IL-1β and IL-18 in serum of rats in TFPF high- and medium-dose groups could be decreased to different degrees （P<0.05， P<0.01）， while the content of MCP-1 in TFPF high-dose group was significantly decreased （P<0.01）. The content of TGF-β1 in renal tissue homogenate in TFPF high- and medium-dose groups was significantly decreased （P<0.05， P<0.01）， and the content of IL-1β in renal tissue homogenate in TFPF medium-dose group was significantly decreased （P<0.01）. HE staining showed that each dose group of TFPF could improve the status of renal tubular epithelial cells， reduce cytoplasmic swelling and the number of nuclear pyknosis to varying degrees. The positive rate of TUNEL staining was decreased （P<0.01） and DNA damage was decreased. The expression of NLRP3， Caspase-1， IL-1β and NF-κB protein in renal tissue cells was inhibited （P<0.05， P<0.01）. ConclusionTFPF protects rats from gouty nephropathy by inhibiting the secretion of inflammatory cytokines. Specifically， it may inhibit the activation of NF-κB and NLRP3/Caspase-1 pathways to reduce the expression， maturation， and release of inflammatory cytokines such as IL-1β and IL-18 and further inhibit pyroptosis， thereby reversing the inflammatory injury of kidney in gouty nephropathy.

Blood mRNA Expression Profiles of Autophagy, Apoptosis, and Hypoxia Markers on Blood Cardioplegia and Custodiol Cardioplegia Groups

Abstract Introduction: Blood cardioplegia (BC) and Custodiol cardioplegia (CC) have been used for a long time in open heart surgery and are highly effective solutions. The most controversial issue among these two is whether there is any difference between them regarding myocardial damage after ischemia surgery. In this study, autophagy, apoptosis, and hypoxia markers were investigated and that way we evaluated the differences between BC and CC patients. Methods: A total of 30 patients were included in this study, using two different cardioplegic solutions. Three different whole blood samples of the patients were taken from a central vein (preoperatively, immediately postoperatively, and one day after surgery). Total ribonucleic acid was extracted from these samples. Quantitative real-time polymerase chain reaction was performed, and changes in gene expression were determined by the 2-∆∆Ct method of relative quantification. Results: In the CC group, Beclin gene expression level was found to be higher and this difference was statistically significant (P=0.0024). Similarly, cysteine-aspartic acid protease (caspase) 9 and hypoxia-inducible factor 1α messenger ribonucleic acid (mRNA) gene expression level increased and were significantly different in the CC group. In the BC group, Beclin and microtubule-associated protein light chain 3 expressions were higher in the samples taken one day after surgery. Caspases 3 and 8 gene expressions were significantly different in the BC group. Conclusion: As a result of the analysis performed between the two cardioplegia groups, it has been shown that CC harms the myocardium more than BC at the level of mRNA expression of related markers.

Expression of lncRNA Tsix in a mouse model of N-methyl-D-aspartic acid-induced retinal excitotoxicity and its significance / 中华实验眼科杂志

Objective:To investigate the damage effect of different concentrations of N-methyl-D-aspartic acid (NMDA) to retinal ganglion cells (RGCs) in mice and explore the expression of long noncoding RNA (lncRNA) Tsix in the retina of mice with excitotoxicity as well as the protective effect of lncRNA Tsix on retina and RGCs.Methods:A total of 105 C57B6/J mice at 7-8 weeks of age were selected and randomly divided into the normal control group, 2 mmol/L NMDA group, 10 mmol/L NMDA group, 20 mmol/L NMDA group and 40 mmol/L NMDA group using a random number table method, with 21 mice in each group.In the normal control group, the mice were intravitreally injected with 1 μl of sodium chloride solution in the right eye, and mice were given intravitreal injection of 1 μl of different doses of NMDA according to grouping.At one week after the injection, the thickness of each retinal layer, the number of ganglion cell layer (GCL) cells and the number of RGCs were analysed and compared among different groups through optical coherence tomography (OCT), hematoxylin-eosin staining, retinal whole mount staining and immunofluorescence staining.RNAscope in situ hybridization was used to verify the expression of lncRNA Tsix in the GCL of different groups.The quantitative real-time PCR was used to detect the transcript levels of Tsix in different groups.This study was approved by an Ethics Committee of Tianjin Medical University (No.SYXK2018-0004), and the use of experimental animals was in accordance with the regulations of Tianjin Medical University and ARVO statement. Results:The OCT results showed that the total retinal thickness of mice in the 2, 10, 20 and 40 mmol/L NMDA groups were (255.00±6.63), (252.40±6.41), (248.67±6.20) and (229.11±10.37)μm, respectively, which were thinner than (269.60±20.01)μm in the normal control group, and the differences were statistically significant (all at P<0.05). Hematoxylin-eosin staining showed that the cells in the GCL of the normal control group were uniform and compact, and arranged in a single layer with large and round nuclei.In the NMDA groups, the cells were uneven in volume with vacuoles and nuclear pyknosis.The cell density in the GCL was decreased significantly with the increasing NMDA doses in NMDA groups in comparison with the normal control group, and the differences were statistically significant (all at P<0.05). In the 20 mmol/L NMDA group, the cell density in the GCL was reduced to half of the normal control group.The results of retinal whole mount staining showed that the density of β3-tubulin-positive RGCs was decreased significantly as the dose of NMDA increased in NMDA groups, and the differences were statistically significant compared with the normal control group (all at P<0.05). The number of RGCs in the 10 mmol/L NMDA group was reduced to half of that in the normal control group.RNAscope results showed that lncRNA Tsix was mainly expressed in the cytoplasm of the GCL cells.The proportion of lncRNA Tsix-positive cells was significantly reduced with the increase of the NMDA dose ( F=13.670, P<0.01). The quantitative real-time PCR results verified that the trend of Tsix expression was consistent with the RNAscope result. Conclusions:NMDA exerts a dose-dependent damage to the layer thickness of mouse retina and RGCs.The expression of lncRNA Tsix in mouse retina is mainly enriched in the cytoplasm of the cells in the GCL, and the transcript level of Tsix is reduced with the increase of NMDA concentration and have a protective effect on RGCs.

Jinxiangdan Inhibits NLRP3/IL-1β/Caspase-1 Pathway to Alleviate Myocardial Ischemia-reperfusion Injury in Rats / 中国实验方剂学杂志

Objective:To observe the effect of Jinxiangdan （JXD） on NOD-like receptor pyrin domain-containing-3 （NLRP3）/cysteine-dependent aspartate-directed protease-1 （Caspase-1）/interleukin-1<italic>β</italic> （IL-1<italic>β</italic>） signaling pathway in myocardium of rats with myocardial ischemia-reperfusion injury （MIRI） and explore the protective effect and mechanism of JXD against MIRI. Method:Fifty male SD rats were randomly divided into the sham operation group， model group， high- and low-dose JXD groups， and positive drug （Di'ao Xinxuekang） group， with 10 rats in each group. Seven days before modeling， rats in the JXD groups were separately treated with intragastric administration of 0.72 and 0.18 g·kg^-1 JXD tablets， the ones in the sham operation group and model group with the same volume of normal saline， and those in the positive drug group with 1.29 g·kg^-1 Di'ao Xinxuekang. Twelve hours after the last intragastric administration， the anterior descending branch of the left coronary artery was ligated for 30 min and then re-perfused for 60 min for inducing MIRI. ST segment elevation was detected by electrocardiogram（ECG） for model evaluation. The contents of creatine kinase （CK） and lactate dehydrogenase （LDH） in cardiac tissue were measured by colorimetry. Hematoxylin-eosin （HE） staining was conducted for observing myocardial histopathological changes， followed by the detection of cardiomyocyte apoptosis by DNA in situ nick end-labeling （TUNEL） assay. The protein and mRNA expression levels of NLRP3， Caspase-1， and IL-1<italic>β</italic> were detected by Western blot and real-time polymerase chain reaction （Real-time PCR）， respectively. Result:Compared with sham operation group， the model group exhibited obviously elevated ST segment （<italic>P</italic><0.01）， enhanced CK and LDH activities in the myocardium （<italic>P</italic><0.01）， increased apoptotic cardiomyocytes （<italic>P</italic><0.01）， and up-regulated NLRP3， Caspase-1， and IL-1<italic>β</italic> protein and mRNA expression （<italic>P</italic><0.01）. Compared with model group， JXD at both the high and low doses and Di'ao Xinxuekang significantly lowered the ST segment （<italic>P</italic><0.05，<italic>P</italic><0.01）， diminished the CK and LDH activities in myocardial tissue （<italic>P</italic><0.05，<italic>P</italic><0.01）， improved the apoptosis of cardiomyocytes （<italic>P</italic><0.05，<italic>P</italic><0.01）， and down-regulated the mRNA and protein expression levels of NLRP3， Caspase-1， and IL-1<italic>β</italic> in myocardial tissue （<italic>P</italic><0.05，<italic>P</italic><0.01）. The ST segment of ECG in the low-dose JXD group was increased as compared with that in the Di'ao Xinxuekang group （<italic>P</italic><0.05）， while the ST segment in the high-dose JXD group was obviously elevated （<italic>P</italic><0.05）. Besides， the green fluorescence intensities in the low- and high-dose JXD groups and the Di'ao Xinxuekang group remarkably declined （<italic>P</italic><0.05，<italic>P</italic><0.01）. The mRNA and protein expression levels of NLRP3， Caspase-1， and IL-1<italic>β</italic> in the high-dose JXD group were down-regulated （<italic>P</italic><0.05）. Conclusion:JXD alleviates MIRI possibly by lowering NLRP3 and IL-1<italic>β</italic> expression and inhibiting cardiomyocyte apoptosis.

Impacts of electroacupuncture on neurological function and protein expressions of apoptosis-related Cyt-C and Caspase-9 in rats with traumatic brain injury / 中国针灸

OBJECTIVE@#To observe the impacts of electroacupuncture (EA) on neurological function, the pathological morphology in brain tissue, apoptosis level and the protein expressions of apoptosis-related cytochrome C (Cyt-C) and cysteine aspartic acid protease-9 (Caspase-9) in the rats with traumatic brain injury (TBI) and explore the potential mechanism of EA in treatment of TBI.@*METHODS@#A total of 70 clean-grade SD mice were randomized into a blank group (8 rats), a sham-operation group (8 rats), a model group (27 rats) and an EA group (27 rats). In terms of interventions of 3, 7 and 14 days, 3 subgroups were divided in the model group and the EA group successively, 9 rats in each subgroup. The modified Feeney free-fall percussion method was adopted to establish TBI models of rats. In the sham-operation group, only the skull was exposed and drilled and no free-fall percussion was exerted. One day after modeling, EA was given in the rats of EA group at "Shuigou" (GV 26), "Baihui" (GV 20) and "Neiguan" (PC 6) and "Zusanli" (ST 36) on the affected side, with intermittent wave, 2 Hz in frequency, once daily, 10 min each time, for 3, 7 and 14 days successively. Separately, on the day 3, 7 and 14 of intervention, the modified neurological severity scale (mNSS) was used to evaluate the degree of neurological function injury in the rats, HE staining and Nissl staining were to observe the pathological and morphological changes in brain tissue, TUNEL method was to observe the level of apoptosis in brain tissue and immunohistochemistry (IHC) method and Western blot were to determine the protein expressions of Cyt-C and Caspase-9 in brain tissue.@*RESULTS@#Compared with the sham-operation group, on the day 3, 7 and 14 of intervention, mNSS scores were increased obviously in the rats of the model group respectively (<0.01). Compared with the model group, on the day 3, 7 and 14 of intervention, mNSS scores were reduced in the rats of the EA group respectively (<0.05). On day 3 of intervention, in brain injury region of the rats in the model group and the EA group, gross tissue necrosis, nuclear fragmentation, consolidation and obvious vacuolar changes, reduced Nissl bodies and scattered arrangement were found. On day 7 and 14 of intervention, in the model group and the EA group, the new connective tissue filling and normal cells were visible and Nissl bodies increased. The overall repair and Nissl body quantity in the EA group were better than the model group. Compared with the sham-operation group, on day 3, 7 and 14 of intervention, the numbers of apoptotic cells were increased obviously in the model group (<0.01) and they were reduced in the EA group as compared with the model group (<0.05). Compared with the sham-operation group, on day 3, 7 and 14 of intervention, the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue were all increased obviously in the model group (<0.01) and they were all reduced in the EA group as compared with the model group successively (<0.05).@*CONCLUSION@#Electroacupuncture remarkably improves the condition in the neurological function injury and reduces apoptosis degree in TBI model rats, which is likely related to the down-regulation of the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue and further to bring the impacts on mitochondria mediated apoptosis process.

Effects of N-methyl-D-aspartate Receptor in Keratinocyte on TypeⅠComplex Regional Pain Syndrome / 中国医学科学院学报

To observe the cell origin of N-methyl-D-aspartic acid(NMDA)receptor expression in skin after chronic ischemic pain modeling in rats and explore the role of NMDA receptor in type Ⅰ complex regional pain syndrome. Forty-two adult male Sprague-Dawley rats were randomly divided into five groups:sham operation group(=12),chronic post ischemia pain(CPIP)group(=12),CPIP+normal saline(NS)group(=6),CPIP+NMDA group(=6),and CPIP+MK801 group(=6).Six rats in the sham operation group and CPIP group were sacrificed under deep anesthesia one day after modeling.The plantar skin and L3-L5 spinal cord tissue were used for NR1(NMDA receptor)subunit immunofluorescence detection and for Western blotting of NR1,interleukin(IL)-1β,and tumor necrosis factor(TNF)-α.For the remaining rats,the mechanical withdrawal threshold(MWT)values on the 2nd,6th,10th and 14th day after ischemia were recorded,and the corresponding drugs were injected subcutaneously from the 6th day after ischemia.The skin and L3-L5 spinal cords were collected on the 14th day,and the same detection methods were applied. Compared with the sham operation group,the CPIP group had significantly higher expressions of NR1(1.708±0.064;=12.120, <0.001),IL-1β(2.575±0.305;=5.158, =0.003),and TNF-α(2.691±0.217;=7.786, <0.001)in the skin on the first day after modeling.After intervention with NMDA and MK801,the MWT value was [(20.37±0.95)g] in the CPIP+NS group,which was significantly higher than that in CPIP+NMDA group [(15.85±1.09)g;=10.920, <0.001] but significantly lower than that in CPIP+MK801 group[(22.95±0.96)g;=6.421, <0.001] 10 days after modeling.On the 14th day,compared with the MWT of the CPIP+NS group [(21.57±0.96)g],the CPIP+NMDA group had significantly decreased MWT value [(16.53±1.63)g;=12.190, <0.001],and the CPIP+MK801 group had significantly increased MWT value [(23.27±1.28)g;=4.094, =0.025].Compared with the sham operation group,the CPIP group had significantly increased NR1 expression(1.708±0.064;=10.910, <0.001)and the CPIP+NS group had significantly increased expressions of IL-1β(2.518±0.147;=11.010, <0.001)and TNF-α(1.949±0.184;=10.870, <0.001).Compared with the CPIP+NS group,the CPIP+NMDA group had significantly increased expressions of IL-1β(4.816±0.607;=16.670, =0.003)and TNF-α(2.629±0.349;=7.790, <0.001)and the CPIP+MK801 group had significantly decreased expressions of IL-1β(1.048±0.257;=10.660, =0.003)and TNF-α(0.790±0.165;=13.280, <0.001). NMDA receptor activation in skin keratinocytes after chronic ischemia in rats hinders the expression of inflammatory cytokines such as IL-1β and TNF-α,which may be involved in central sensitization and pain conduction of type Ⅰ complex regional pain syndrome.

Effect of Anmeidan in Improving Learning and Memory Levels of Sleep Deprived Rats Through Mitochondrial Mediated Hippocampal Neuronal Apoptosis / 中国实验方剂学杂志

Objective:To explore the effect of Anmeidan （AMD） on the learning and memory levels of sleep deprived rats through mitochondrial mediated hippocampal neuronal apoptosis pathway. Method:Forty-eight SD rats were randomly divided into blank group， model group， low， medium， high-dose AMD groups （4.86， 9.72， 19.44 g·kg-1·d-1） and estazolam group （0.1 mg·kg-1·d-1）. Insomnia model was prepared by self-made sleep deprivation box for 14 days. Morris water maze was used to detect learning and memory levels， enzyme-linked immunosorbent assay （ELISA） was used to detect the expressions of cytochrome C （Cyt-C）， cysteine aspartic acid protease-3 （Caspase-3） in hippocampus. Transmission electron microscopy （TEM） was used to observe the morphological structure of mitochondria in hippocampus. Protein and mRNA expressions of Cyt-C， Caspase-3， Bcl-2， Bax were detected by immunofluorescence （IF） and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） respectively. Result:In the model group， the incubation period of the platform and the total distance of swimming and the time of first arriving platform were prolonged， the number of platform crossing and the time of target quadrant movement were reduced， protein and mRNA expressions of Bcl-2 dropped， protein and mRNA expressions of Bax increased （P<0.01）， and mitochondrial structure was abnormal with crista fracture， swelling and deformation. And protein and mRNA expressions of Cyt-C， Caspase-3 increased significantly （P<0.01）. Low， medium and high-dose AMD groups could improve levels of space exploration and navigation of SD rats （P<0.01）， increase protein and mRNA expressions of Bcl-2， decrease protein and mRNA expressions of Bax， improve the damage of mitochondria， and decrease the protein and mRNA expressions of Cyt-C， Caspase-3 （P<0.01）. Conclusion:AMD can improve the learning and memory levels of SD rats， the effect is related to the mitochondrial mediated hippocampal neuronal apoptosis pathway and decrease of Cyt-C and Caspase-3 expressions.

Effect of H2S on the expression of p38MAPK and caspase3 and apoptosis in renal tissue of urogenous sepsis / 医学研究生学报

ObjectiveTo study the mechanism of renal injury in urogenic sepsis and explore the effect of H2S on the expression of p38 mitogen-activated protein kinase (p38MAPK) and Cysteine protease 3(Caspase3) and apoptosis in renal tissue of urogenic sepsis. Hydrogen sulfide (H2S) has a wide range of biological effects and has a certain protective effect on the kidney. The main objective of this study is to investigate the effect of H2S on the expression of p38 mitogen-activated protein kinase (p38MAPK), cysteine proteinase 3 (Caspase3), and cell apoptosis in renal tissue of urogenic sepsis, and further to study the mechanism of urinary sepsis renal injury.MethodsNew Zealand rabbits (n=40) were divided into five groups Control, Sham, Sepsis, NaHS, and PAG, with eight rabbits in each group. The vital signs, blood routine white blood cell count, neutrophil count, and renal function (Cr, BUN) of five groups of New Zealand rabbits were recorded before the operation, 24 hrs after the operation, 48 hrs after the operation, and 72 hrs after the operation. 72 hrs after the operation, the left kidney tissue was taken for HE staining to observe the changes of cell morphology and structure of the kidney tissue. The apoptotic cells in renal tissue were labeled by Tunel assay (in situ terminal transferase labeling technique). The expression levels of p38MAPK and Caspase3 in renal tissue were tested by ELISA.Results The apoptosis indexes of renal tissue cells in group Control and group Sham were (5.65±2.43)% and (5.57±2.72)%, respectively, with no statistically significant difference (P>0.05). The apoptosis index of rabbit kidney tissue in group Sepsis was (25.26±2.70)%, which was significantly higher than that in group Control and group Sham (P0.05). Pairwise comparison between groups Sepsis, NaHS, and PAG showed statistically significant differences (P<0.05). The expression levels of group Sepsis and group PAG were significantly higher than that of group NaHS (P<0.05). The expression level of group PAG was significantly higher than that of group Sepsis (P<0.05).Conclusion H2S can alleviate renal damage caused by urine-derived sepsis, and its mechanism combined with H2S to suppress the expression of p38MAPK and Caspase3 in the renal tissue of urogenous sepsis, thereby reducing renal cell apoptosis Death.

Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.

Identification of Gly/NMDA receptor Antagonist from Chromolaena odorata’s Derived compounds using Induced Fit Docking and ADME study

The ionotropic activation of N-methyl-D-aspartic acid (NMDA) plays asignificant role in different type of neurodegenerative disease, as it is a tetramer withtwo Glycine binding subunit and two glutamate subunits. NMDA receptor can beinhibited by either blocking of the glycine site or glutamate site. Previously reportedinhibitors of NMDA receptor focus on the inhibition of the glutamate subunit, which wasreported to be associated with side effects such as ataxia, memory deficits, andneurotoxicity. Therefore, different compounds with antagonistic effect are beenexplored on Gly/NMDA site. Glide XP docking was employed in screening phytoconstituent of Chromolaena odorata against Gly/NMDA receptor for hit compounds withantagonistic properties. The hit compounds were further subjected to Induced fitdocking (IFD) and Lipinski rule of five. The final selection was based on Rigid XP dockingscore using co-crystallized ligand as threshold docking score, interaction with receptorsite residues, and IFD score. Ferulic acid, caffeic acid and scutellarein recorded bindingaffinity of -8.752Kcal/mol, 10.004 Kcal/mol and -9.096 Kcal/mol respectively, which ishigher than the binding affinity of co-crystallized ligand. Induced fit score obtained were-614.38, -614.03 and -616.31 for ferulic acid, caffeic acid and scutellarein respectively.The result obtained in this study shows the potency of phytochemical from C. odorata toinhibit NMDA receptor. ADME study showed that the drug-like nature of thesecompounds.

Effect of Modified Si Junzitang Drug Serum on Expression of Apoptosis-related Molecules of Gastric Cancer Cell SGC-7901 / 中国实验方剂学杂志

Objective: To explore the effect of modified Si Junzitang (MSJZT) drug serum on the expression of apoptosis-related molecules of gastric cancer cell SGC-7901 and further its anti-tumor mechanism.Method: A total of 40 SD rats were randomly divided into four groups:low-dose,middle-dose,high-dose MSJZT (0.213,0.426,0.853 g·kg-1) groups and normal group (n=10).The treatment groups were administrated through gastric perfusion,and the normal group was given the equivalent volume of normal saline for 10 days.1.5 h after the last treatment,chloral hydrate peritoneal anesthesia was performed,blood was collected from heart,and different doses of serum were separated to prepare drug-containing serum of low-dose,middle-dose,high-dose MSJZT groups,in order to incubate SGC-7901 gastric cancer cell.Early and late apoptosis rates were detected with flow cytometry.Afterwards,the tumor suppressor gene p53,c-nucleoprotein gene (c-Myc),cysteine-aspartic acid protease-3(Caspase-3),B-cell lymphoma-2(Bcl-2) mRNA expressions were confirmed by fluorescence quantitative polymerase chain reaction (Real-time PCR).The protein expressions of p53,c-Myc,Caspase-3,Bcl-2 were detected by immunofluorescence.Result: Compared with the normal group,the high-dose MSJZT group could obviously increase the apoptosis rate to 22.58%(PPPPPPConclusion: MSJZT drug serum could exert an anti-tumor effect by inhibiting the expression of the anti-apoptotic protein Bcl-2,and promoting the expressions of pro-apoptotic-related molecules p53,c-Myc,Caspase-3.

Risk Assessment of "Other Substances" – L-aspartic Acid

The Norwegian Scientific Committee for Food Safety (Vitenskapskomiteen for mattrygghet, VKM) has, at the request of the Norwegian Food Safety Authority (Mattilsynet; NFSA), assessed the risk of "other substances" in food supplements and energy drinks sold in Norway. VKM has assessed the risk of doses in food supplements and concentrations in energy drinks given by NFSA. These risk assessments will provide NFSA with the scientific basis while regulating the addition of "other substances" to food supplements and other foods. "Other substances" are described in the food supplement directive 2002/46/EC as substances other than vitamins or minerals that have a nutritional and/or physiological effect. It is added mainly to food supplements, but also to energy drinks and other foods. VKM has not in this series of risk assessments of "other substances" evaluated any claimed beneficial effects from these substances, only possible adverse effects. The present report is a risk assessment of specified doses of L-aspartic acid in food supplements, and it is based on previous risk assessments and articles retrieved from literature searches. According to information from NFSA, L-aspartic acid is an ingredient in food supplements sold in Norway. NFSA has requested a risk assessment of 3000, 3500, 4000, 4500, 5000 and 5700 mg/day of L-aspartic acid in food supplements. L-aspartic acid is a dispensable dicarboxylic amino acid that can be produced by the transamination of oxaloacetic acid, an intermediate in the metabolism of e.g. glucose and some amino acids. L-aspartic acid is present in frequently consumed foods of animal and plant origin and is also a component of the sweetener aspartame. Dietary intake of aspartic acid in Norway is not known, but data from NHANES III (USA) suggest a mean dietary intake of about 6.5 g/day in adults. The highest intake was seen in men 31 through 50 years of age at the 99th percentile of 15.4 g/day. In the literature review we did not identify any long-term studies in human individuals that could be used for risk assessment. Short-term human studies found no adverse health effect when L-aspartic acid was given in acute doses ranging from 1 to 10 g/day, for time periods between one single dose and four weeks. None of these studies were undertaken to assess the toxicity of L-aspartic acid. In the literature search, two animal studies were identified of which one was a 90-day subchronic toxicity study. In that study, a no observed adverse effect level (NOAEL) of 697 mg/kg bw per day in male rats and 715 mg/kg bw per day in female rats was established. No neurotoxicity was found, however a toxic effect on kidneys and possibly salivary glands was observed at 1400 mg/kg bw per day (lowest observed adverse effect level, LOAEL). For the risk characterisation, the NOAEL of 697 mg/kg bw per day derived from the abovementioned subchronic toxicity study in rats was used for comparison with the estimated exposures from food supplements. The calculated Margin of Exposure (MOE) values for this NOAEL ranged from 5 to 16 for a daily intake of 3000-5700 mg/day of Laspartic acid. These low MOE-values may not be regarded as acceptable since L-aspartic acid has caused toxic effects on the kidneys (regenerative renal tubules with tubular dilation) and acinar cell hypertrophy of salivary glands in rats. Further, direct information regarding potential adverse health effects in humans is not available due to absence of long-term studies. In adults (≥18 years), adolescents (14 to < 18 years) and children (10 to < 14 years), the specified doses 3000, 3500, 4000, 4500, 5000 and 5700 mg/day L-aspartic acid in food supplements may represent a risk of adverse health effects. Children younger than 10 years were not within the scope of the present risk assessment.

Development of a UPLC-MS/MS method for the therapeutic monitoring of L-asparaginase

This study aimed to develop a UPLC-MS/MS method for determining plasma levels of L-aspartic acid and L-asparagine and the activity of L-asparaginase. L-aspartic acid, L-asparagine, and L-aspartic acid-2,3,3-d3 were extracted from human plasma by protein precipitation with sulfosalicylic acid (30%, v/v). The plasma samples were analyzed using an Imtakt Intrada amino acid analysis column with 25 mM ammonium formate and 0.5% formic acid in acetonitrile as the mobile phase with step gradient method at a flow rate of 0.5 mL/min. The injection volume was 5 µL, and the total run time was 15 min. Inter- and intra-batch accuracies (%) ranged from 96.62–106.0% for L-aspartic acid and 89.85–104.8%, for L-asparagine, and the coefficient of variation (CV%) did not exceed 7%. The validation results for L-aspartic acid and L-asparagine satisfied the specified criterion, however, the results for L-asparaginase activity assay showed a borderline validity. This study could be a foundation for further development of therapeutic drug monitoring systems using UPLC-MS/MS.

Effects of 17β-estradiol on threshold of rat neuropathic pain / 重庆医学

Objective To investigate the effect of 17β-estradiol on the pain threshold in neuropathic rats.Methods Fifty fe-male SD rats were divided into five groups by adopting the random number table method,the sham operation group(separating and exposing the left sciatic nerve without ligation and giving the saline injection),the other four groups were established as chronic compression injury(CCI)model of sciatic nerve:CCI group(saline injection),estradiol group(17β-estradiol injection),antagonist group(AP-5 injection)and composite group(17β-estradiol and AP-5 injection),10 cases in each group.The rat mechanical pain threshold value was examined by adopting the paw withdrawal mechanical threshold(PWMT),the rat thermal threshold was exam-ined by adopting the paw withdrawal thermal latency(PWTL),and the expression of NMDAR1 protein was determined by immuno-histochemistry and Western blot.Results Compared with the sham operation group,PWM T in the CCI group and estradiol group was decreased(P<0.05)and PWTL was shortened(P< 0.05),while the expression of NMDAR1 protein was significantly in-creased(P<0.05).Compared with the CCI group,PWMT in the estradiol group was decreased(P<0.05)and PWTL was short-ened(P<0.05),while the expression of NMDAR1 protein was significantly increased(P<0.05).Conclusion 17β-estradiol can de-crease the pain threshold of the neuropathic pain rats by increasing the NMDAR1 expression.