Investigation on the pharmacological mechanisms of Shirebi granules in treating gouty arthritis with damp-heat syndrome based on crosstalk of Wnt/β-catenin signaling / 药学学报

Gouty arthritis is a type of metabolic rheumatic disease caused by autoimmune abnormalities. Currently, the use of Western medicine in the clinical treatment of gouty arthritis has been associated with a high risk of adverse reactions. Therefore, there is a growing interest in exploring therapeutic drugs from traditional Chinese medicine as a potential alternative. According to the theory of traditional Chinese medicine, gouty arthritis has been classified as damp-heat arthralgia syndrome. Shirebi granules has been found to have good clinical efficacy in treating gouty arthritis. However, its underlying pharmacological mechanisms remain unclear. To address this problem, the study first established the interaction network of candidate targets for Shirebi granules, which is used to treat damp-heat syndrome of gouty arthritis. Then, the key candidate targets of Shirebi granules for treating gouty arthritis with damp-heat syndrome were screened by calculating the topological features of the network nodes. Then, the functional mining of the key candidate targets revealed that the candidate targets of Shirebi granules may intervene in the biological process of inflammatory response and lipid metabolism through the crosstalk of Wnt/β-catenin signaling. To verify the effectiveness of Shirebi granules in treating gouty arthritis with damp-heat syndrome, a rat model was established. The results demonstrated that the granules significantly improved the severity of arthritis in rats with this condition, reduced joint inflammation, gait score, swelling index, increased mechanical pain threshold (P < 0.05), and reduced the content of serum inflammatory factors IL-1β, IL-6, and TNF-α in gouty arthritis rats with damp-heat syndrome (P < 0.01) gouty. It was also found that Shirebi granules effectively alleviated the symptoms of dampness heat syndrome such as local joint fever and dry mouth by reducing the temperature of the joints in acute gouty arthritis with damp-heat syndrome (AD) rats, increasing the threshold of heat pain, increasing water intake (P < 0.01), and inhibiting abnormal changes in the content of fatty acid oxidation related enzymes (P < 0.01). Western blot analysis showed that Shirebi granules increased the protein expression levels of Wnt and β-catenin (P < 0.01) while decreasing the protein expression of p65, p-p65 and PPARγ (P < 0.01) in rats with gouty arthritis and damp-heat syndrome. The results showed that Shirebi granules may reverse the "inflammation-immune" imbalance and lipid metabolism disorder by regulating the crosstalk of Wnt/β-catenin signaling, and play a role in alleviating the severity of the disease. This study provides a methodological reference for elucidating the pharmacological mechanisms of traditional Chinese medicine formulas. It also presents research ideas for the appropriate clinical use of Chinese patent medicines and the development of new clinical drugs for gouty arthritis therapy. The animal welfare and experiment procedures of this study were performed in accordance with the regulations of the Experimental Animal Ethics Committee of Experimental Research Center, China Academy of Chinese Medical Sciences (grant No. ERCCACMS11-2302-08).

Comparison of the Nerve Regeneration Capacity and Characteristics between Sciatic Nerve Crush and Transection Injury Models in Rats / 生物医学与环境科学（英文）

OBJECTIVE@#To provide useful information for selecting the most appropriate peripheral nerve injury model for different research purposes in nerve injury and repair studies, and to compare nerve regeneration capacity and characteristics between them.@*METHODS@#Sixty adult SD rats were randomly divided into two groups and underwent crush injury alone (group A, n = 30) or transection injury followed by surgical repair (group B, n = 30) of the right hind paw. Each group was subjected to the CatWalk test, gastrocnemius muscle evaluation, pain threshold measurement, electrophysiological examination, retrograde neuronal labeling, and quantification of nerve regeneration before and 7, 14, 21, and 28 days after injury.@*RESULTS@#Gait analysis showed that the recovery speed in group A was significantly faster than that in group B at 14 days. At 21 days, the compound muscle action potential of the gastrocnemius muscle in group A was significantly higher than that in group B, and the number of labeled motor neurons in group B was lower than that in group A. The number of new myelin sheaths and the g-ratio were higher in group A than in group B. There was a 7-day time difference in the regeneration rate between the two injury groups.@*CONCLUSION@#The regeneration of nerve fibers was rapid after crush nerve injury, whereas the transection injury was relatively slow, which provides some ideas for the selection of clinical research models.

Baimai Ointment relieves chronic pain induced by chronic compression of dorsal root ganglion in rats by regulating neuroactive ligand-receptor interaction and HIF-1 signaling pathway / 中国中药杂志

The Baimai Ointment with the effect of relaxing sinew and activating collaterals demonstrates a definite effect on Baimai disease with pain, spasm, stiffness and other symptoms, while the pharmacodynamic characteristics and mechanism of this agent remain unclear. In this study, a rat model of chronic compression of L4 dorsal root ganglion(CCD) was established by lumbar disc herniation, and the efficacy and mechanism of Baimai Ointment in the treatment of CCD were preliminarily explored by behavioral tests, side effect evaluation, network analysis, antagonist and molecular biology verification. The pharmacodynamic experiment indicated that Baimai Ointment significantly improved the pain thresholds(mechanical pain, thermal pain, and cold pain) and gait behavior of CCD model rats without causing tolerance or obvious toxic and side effects. Baimai Ointment inhibited the second-phase nociceptive response of mice in the formalin test, increased the hot plate threshold of normal mice, and down-regulated the expression of inflammatory cytokines in the spinal cord. Network analysis showed that Baimai Ointment had synergistic effect in the treatment of CCD and was related to descending inhibition/facilitation system and neuroinflammation. Furthermore, behavioral tests, Western blot, and immunofluorescence assay revealed that the pain-relieving effect of Baimai Ointment on CCD may be related to the regulation of the interaction between neuroactive ligand and receptors(neuroligands) such as CHRNA7, ADRA2A, and ADRB2, and the down-regulation of the expression of NOS2/pERK/PI3K, the core regulatory element of HIF-1 signaling pathway in spinal microglia. The findings preliminarily reveal the mechanism of relaxing sinew and activating collaterals of Baimai Ointment in the treatment of Baimai disease, providing a reference for the rational drug use and further research of this agent.

Mechanism of Mongolian drug Naru-3 in initiation of neuroinflammation of neuropathic pain from MMP9/IL-1β signaling pathway / 中国中药杂志

Neuropathic pain(NP) has similar phenotypes but different sequential neuroinflammatory mechanisms in the pathological process. It is of great significance to inhibit the initiation of neuroinflammation, which has become a new direction of NP treatment and drug development in recent years. Mongolian drug Naru-3 is clinically effective in the treatment of trigeminal neuralgia, sciatica, and other NPs in a short time, but its pharmacodynamic characteristics and mechanism of analgesia are still unclear. In this study, a spinal nerve ligation(SNL) model simulating clinical peripheral nerve injury was established and the efficacy and mechanism of Naru-3 in the treatment of NPs was discussed by means of behavioral detection, side effect evaluation, network analysis, and experimental verification. Pharmacodynamic results showed that Naru-3 increased the basic pain sensitivity threshold(mechanical hyperalgesia and thermal radiation hyperalgesia) in the initiation of SNL in animals and relieved spontaneous pain, however, there was no significant effect on the basic pain sensitivity threshold and motor coordination function of normal animals under physiological and pathological conditions. Meanwhile, the results of primary screening of target tissues showed that Naru-3 inhibited the second phase of injury-induced nociceptive response of formalin test in mice and reduced the expression of inflammatory factors in the spinal cord. Network analysis discovered that Naru-3 had synergy in the treatment of NP, and its mechanism was associated with core targets such as matrix metalloproteinase-9(MMP9) and interleukin-1β(IL-1β). The experiment further took the dorsal root ganglion(DRG) and the stage of patho-logical spinal cord as the research objects, focusing on the core targets of inducing microglial neuroinflammation. By means of Western blot, immunofluorescence, agonists, antagonists, behavior, etc., the mechanism of Naru-3 in exerting NP analgesia may be related to the negative regulation of the MMP9/IL-1β signaling pathway-mediated microglia p38/IL-1β inflammatory loop in the activation phase. The relevant research enriches the biological connotation of Naru-3 in the treatment of NP and provides references for clinical rational drug use.

An exploration into the therapeutic effects and molecular mechanisms of paeoniflorin in the treatment of adjuvant-induced arthritis rats by a network pharmacology-based research strategy / 药学学报

Paeoniflorin (PAE), the major active compounds of Chinese herbs Radix Paeoniae Alba andChinese patent drug "Total Glucosides of Paeony Capsules", which is effective in the treatment of rheumatoid arthritis (RA), exerted multi-pharmacological activities, such as anti-inflammatory, immune-regulatory, etc. However, its potential action mechanisms remain unclear. Herein, we predicted the putative targets of Radix Paeoniae Alba and constructed an interaction network of putative targets of Radix Paeoniae Alba and known RA-related genes. A list of key putative targets was identified by calculating their topological features (degree, node betweenness and closeness) in the above pharmacological network. Importantly, pathway enrichment analysis revealed that these key putative targets were significantly enriched in several RA-related pathways, including cartilage damage-related IL1B-TNF-TLR2-JUN-MMP1-MMP3 signaling pathway. Further molecular docking simulation showed that PAE, the major active compounds of Radix Paeoniae Alba, has strong binding affinity with MMP1 and MMP3 proteins. Next, in vivo experiments based on the adjuvant-induced arthritis (AIA) animal models showed that PAE significantly alleviated the disease severity and the syndromes of severe redness or swelling in hind limbs of AIA rats, including decreasing the arthritis score, the diameter of the limbs, and elevating body weight and pain thresholds (all P<0.05). ELISA assay indicated that PAE obviously suppressed the abnormal up-regulation of serum inflammatory factors including IL-1β, TNF-α, IL-6, IL-17 and IFN-γ in AIA rats (all P<0.001). Western blot analysis found that PAE simultaneously modulated the abnormal up-regulation of MMP1 and MMP3 proteins in the ankle tissues of AIA rats (all P<0.001) (all procedures in the current study were performed in accordance with the ethical standards of the Center for Laboratory Animal Care, China Academy of Chinese Medical Sciences). In conclusion, PAE alleviated the cartilage damage and disease severity in the progressive process of RA via regulating the IL1B-TNF-TLR2-JUN-MMP1-MMP3 pathway. This study provided the theoretical basis of the PAE for its immune-regulatory effects, and as well provided references for the action mechanism study of extract compounds of Chinese herbs.

Investigation on the inflammation network mechanisms of Wutou decoction acting on neuropathic pain / 药学学报

Wu-tou decoction (WTD) was originally recorded in the synopsis of the golden chamber and it had been widely used for the treatment of neuropathic pain (NP) with exact therapeutic efficacy. However, the underlying molecular mechanisms still remain unclarified. Thus, in this research, we aimed at clarifying the underlying molecular mechanisms of WTD against NP by combining network analysis and experimental validation based on the spinal nerve ligation (SNL) model. Firstly, the network analysis indicated that key targets of WTD were significantly involved in the MAPK signaling pathway (P = 4.04E-12) and four important components of the above pathway, AKT kinase (AKT), MAP kinase kinase 4 (MKK4), c-Jun N-terminal kinase (JNK) and transcription factor AP-1 (JUN) had been reported to play a vital role in neuroinflammation during the disease process of NP. Then, experimental validation results proved that WTD markedly reduce the severity of mechanical allodynia (P<0.01) and cold hypersensitivity (P<0.05) of SNL rats. In addition, Western blot results provided evidence that the phosphorylated protein expression levels of AKT, MKK4, JNK and JUN in the superficial lamina of spinal cord of SNL rats were markedly increased (P<0.001), and WTD could improve the phosphorylated protein expression level of AKT (P<0.001) which was reported to be nerve protective and attenuate the phosphorylated protein expression levels of MKK4, JNK and JUN (P<0.01) which were closely involved into neuroinflammation. In conclusion, this study indicated that WTD might exert anti-hyperalgesia action through the inhibition of neuroinflammation mediated by AKT-MKK4-JNK-JUN which belong to the MAPK signaling pathway. These findings also provided scientific evidences that WTD might be a promising candidate for NP. Animal experiments in this study were approved by the Ethics Committee of Experimental Animals of the China Academy of Chinese Medical Sciences.

Identification of 23 unknown Li minority medicinal plants based on DNA barcoding / 中国中药杂志

DNA barcode molecular biological technique is used to identify the species of 23 unknown Li minority medicinal plants.DNA was extracted from 23 unknown medicines using the Plant Genomic DNA Extraction kit. The ITS2 and psbA-trnH regions were amplified and sequenced bi-directionally. The Codon Code Aligner V 7. 0. 1 was used to proofread and assemble the contigs and generated consensus sequences. All the sequences were submitted to Traditional Chinese Medicine DNA Barcode Database and NCBI Gen Bank to get information of the species identifications. If the maximum similarity of the identification result is ≥ 97%,exact species can be known. If it is between 97% and 90%,samples' genus can be confirmed; If it is <90%,then we can only confirm its family. Finally there are 17 samples can be identified to species level,5 can be identified to genus level and 1 can be identified to family level. This shows that DNA barcoding used in medicinal plants molecular identification,can identify unknown species rapidly and accurately.

Identification of biomarkers to response of Tripterygium Glycosides Tablets acting on rheumatoid arthritis by integrating transcriptional data mining and biomolecular network analysis / 中国中药杂志

Growing clinical evidence shows that a partial rheumatoid arthritis( RA) patient treated with Tripterygium Glycosides Tablets( TGT) may fail to achieve clinical improvement. It is of great clinical significance to predict the therapeutic effect of TGT in RA. Therefore,the aim of the current study was to identify potential biomarkers for TGT treatment in RA. Affymetrix EG1.0 arrays were applied to detect gene expression in peripheral blood mononuclear cells obtained from 6 RA patients( 3 responders and 3 non-responders) treated with TGT. By integrating differential expression data analysis and biomolecular network analysis,360 mRNAs( 185 up-regulated and 175 down-regulated) and 24 miRNAs( 7 up-regulated and 17 down-regulated) which were differentially expressed between TGT responder and non-responder groups were identified. A total of 206 candidate target genes for the differentially expressed miRNAs were obtained based on miRanada and Target Scan databases,and then the miRNA target gene coexpression network and miRNA-mediated gene signal transduction network were constructed. Following the network analyses,three candidate miRNAs biomarkers( hsa-miR-4720-5 p,hsa-miR-374 b-5 p,hsa-miR-185-3 p) were identified as candidate biomarkers predicting individual response to TGT. Partialleast-squares( PLS) was applied to construct a model for predicting response to TGT based on the expression levels of the candidate gene biomarkers in RA patients. The five-fold cross-validation showed that the prediction accuracy( ACC) of this PLS-based model efficacy was 100.00%,100.00%,100.00%,66.67% and 66.67% respectively,and all the area under the receiver operating characteristic curve( AUC) were 1.00,indicating the highly predictive efficiency of this PLS-based model. In conclusion,the integrating transcription data mining and biomolecular network investigation show that hsa-mir-4720-5 p,hsa-mir-374 b-5 p and hsa-mir-185-3 p may be candidate biomarkers predicting individual response to TGT. In addition,the PLS model based on the expression levels of these candidate biomarkers may be helpful for the clinical screen of RA patients,which potentially benefit individualized therapy of RA in a daily clinical setting.

A comparative study on the mechanisms of two classical herbal formulae for rheumatoid arthritis applying cold and heat patterns based on target network / 药学学报

Wu-tou decoction (WTD) and Baihu-Guizhi decoction (BHGZD) as described in the Synopsis of the Golden Chamber have been used extensively for the treatment of rheumatoid arthritis (RA) with apparent therapeutic efficacy. However, characteristics of pharmacological effects and their underlying molecular mechanisms have not been fully elucidated due to a lack of appropriate scientific methodology. In the current study, we performed an integrative approach applying gene expression profiling and network analysis to examine the therapeutic effects and molecular mechanisms of WTD and BHGZD based on adjuvant-induced arthritis (AIA) animal model. Results demonstrated that both WTD and BHGZD could relieve the severity of arthritis in AIA rats, while the significant differences were observed in the changes of the withdrawal response scores and latency time of AIA rats treated with WTD and BHGZD. Mechanistically, our network pharmacology-based investigation demonstrated that the major candidate targets of WTD and BHGZD were significantly associated with several inflammation-immune regulatory pathways, such as Toll-like receptor signaling pathway, T cell receptor signaling pathway, cytokine-cytokine receptor interaction, chemokine signaling pathway, B cell receptor signaling pathway, antigen processing and presentation, Fc epsilon RI signaling pathway, natural killer cell mediated cytotoxicity, as well as leukocyte transendothelial migration. In particular, the major candidate targets of WTD were also involved in the regulation of hormone and energy metabolism, which might be imbalanced during RA progression. In conclusion, the current study revealed differences and similarities regarding the effects and network regulatory mechanisms of WTD and BHGZD. These findings may present a scientific basis for elucidation of mechanisms by which WTD and BHGZD alleviates RA.

Classical drug resistance mutations of HepG2.2.15 cells persistently induced by appropriate concentrations of adefovir / 解放军医学杂志

Objective To observe whether the classic drug-resistant mutations can be induced in various concentrations of adefovir (ADV)-treated HepG2.2.15 cells persistently and explore the mechanism for emergence of drug resistance.Methods HepG2.2.15 cells were cultured continually in 12-well plates with medium containing 0,0.01,0.1,1.0μmol/L concentration of ADV,and passaged every 3 days up to the 110th generations.The intracellular and supernatant HBV DNA was extracted every 10 generations.Intracellular HBV cccDNA was amplified by plasmid-safe ATP dependent DNase (PSAD) digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR assay.And the RT region of supernatant HBV DNA was amplified by one-tube nested PCR assay.Then the classic drug-resistant mutations of the RT region of intracellular cccDNA and supernatant HBV DNA were analyzed using direct PCR sequencing combined with clonal sequencing (more than 20 clones/sample).Results HBV DNA stably replicated in ADV-untreated cells (control group).The intracellular total DNA and cccDNA levels,supernatant HBV DNA level decreased continuously with the prolonged ADV culture duration in 0.01 μmol/L and 0.1μmol/L ADV group.Drug resistant mutations were not detected up to the 110th generation in 0.01 μmol/L ADV group;while rtA181V+N236T mutations were detected at the110th generation in 0.1μmol/L ADV group.The 1.0μmol/L ADV group was ceased of culture at the 15th generation due to inhibited cell growth.Conclusion HBV cccDNA exists in HepG2.2.15 cells,and the classical drug-resistant mutations of rtA181V+N236T could be induced by proper concentration of ADV.

HUVECs extraction and UHPLC LTQ Orbitrap analysis for screening vascular endothelium protective components in Kudiezi injection / 中国中药杂志

An effective method has been employed as a tool for screening active components in Kudiezi injection by using cell chromatography and sensitive UHPLC-HR-MSn method. The potential bioactive components in Kudiezi injection could be selectively bound to the HUVECs target cells first. After cell target desensitization and inactivation, the chemical constituents with cell target affinity were identified by LC-MS, so as to screen the possible active components in Kudiezi injection. Based on the accurate mass measurements and the retention time, in total, 9 compounds were tentatively identified and characterized, including 4 sesquiterpene lactones, 3 phenolic acids and 2 flavonoids. HUVECs biospecific extraction coupled with UHPLC-LTQ-Orbitrap analysis could provide a rapid and efficient method for the identification of potential bioactive components in Kudiezi injection, and provide the reference for further research on its effective materials basis.

Study on effect of total coumarins from Urtica dentata on dextran sulfate sodium-induced colitis in mice / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of total coumarins (TC) from Urtica dentata on dextran sulfate sodium (DSS)-induced colitis in mice.</p><p><b>METHOD</b>The colitis model was established by administering DSS. Having been treated with TC, their body weight was determined. Concentrations of IL-6, IL-10, TGF-beta1 and IFN-gamma were monitored by ELISA. Colon samples were collected for the histopathological examination. Western blot was used to detect TLR4 and NF-kappaB protein expression in colonic tissues.</p><p><b>RESULT</b>TCs from U. dentata effectively controlled the body weight loss of mice with colitis, down-regulated the concentration of IL-6 and IFN-gamma and increased the suppressive cytokines IL-10 and TGF-beta1 in the serum. Additionally, TC alleviated the mucosal damage and decreased the expressions of TLR4 and NF-kappaB in colonic tissues.</p><p><b>CONCLUSION</b>TCs from U. dentata shows the anti-inflammatory effect on colitis in mice by reducing the expressions of TLR4 and NF-kappaB in colonic tissues and regulating pro-and anti-inflammatory cytokines.</p>

Reposition of coronary sinus lead dislocation only using pre-shaped stylet and guidewire: a case report / 中华医学杂志(英文版)

Technologies associated with cardiac resynchronization therapy (CRT) devices and lead systems have progressed. However, dislocation after coronary sinus (CS) lead placement continues to be a problem. We reported on the patient treated with CRT, in whom dislocation of CS lead occurred. In the case, we tried to reposition the CS lead without the left heart delivery system only using pre-shaped stylet and guidewire, and the dislocated CS lead could be successfully repositioned by the method. The method of only using a pre-shaped stylet and guidewire is easier than the conventional way, and it can shorten procedure duration and fluoroscopy time, as well as reduce the cost of treatment, but it is not always feasible.

Quantitative analyses of intrahepatic HBV cccDNA and serum HBsAg in 54 patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To quantitatively detect intrahepatic HBV covalently closed circular DNA (cccDNA) and serum HBsAg; and to analyze the relationship between the two parameters and with serum HBV DNA level.</p><p><b>METHODS</b>Intrahepatic cccDNA (copies/cell) was quantitated by plasmid-safe ATP-dependent Danes (PSAD) digestion in combination with rolling circle amplification and gap-spanning selective real-time PCR assay using formalin fixed paraffin-embedded liver biopsy samples. HBsAg was measured by chemiluminescence's reagent manufactured by Abbott Company using sera sampled at time-point of liver biopsy.</p><p><b>RESULTS</b>Intrahepatic cccDNA level was positively correlated with serum HBsAg level (r = 0.459, P < 0.001), but not correlated with serum HBV DNA level. Serum HBsAg level was positively correlated with serum HBV DNA level (r = 0.328, P = 0.015), and reversely correlated with HBV replicative efficiency defined as the ratio of serum HBV DNA to cccDNA (r = -0.373, P = 0.005).</p><p><b>CONCLUSION</b>In patients with chronic hepatitis B, intrahepatic cccDNA level is correlated with serum HBsAg level. The two parameters combined with serum HBV DNA may comprehensively reflect HBV replication activity and help evaluation of antiviral therapeutic efficacy.</p>

Akt-centered amplification loop plays a critical role in vascular endothelial growth factor/stromal cell-derived factor 1-α cross-talk and cardioprotection / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Vascular endothelial growth factor (VEGF) is one of major mediators of angiogenesis and survival factor in some tissue, however, its direct effects on cardiomyocytes remain poorly understood.</p><p><b>METHODS</b>Rat neonatal ventricular myocytes were cultured in vitro. Akt phosphorylation was measured by Western blotting; the expression of stromal cell-derived factor α (SDF-1α)/CXCR4 axis was evaluated by real-time PCR and Western blotting. LY294002 and AMD3100 were used to interfere with the signaling of VEGF and SDF-1α/CXCR4 axis. Cardiac myocytes viability and injury were evaluated by trypan blue staining and lactate dehydrogenase (LDH) release.</p><p><b>RESULTS</b>Treatment of neonatal rat ventricular myocytes with VEGF induced phosphorylation of Akt in a dose and Flk-1 dependent manner. VEGF attenuated H2O2 induced cardiac myocyte death. The phosphoinositol-3-kinase (PI3K) inhibitor, LY294002 and Flk-1 antibody abolished the beneficial effects of VEGF on H2O2 induced cell death. In the mean time SDF-1α-CXCR4 axis was up-regulated by VEGF through PI3K-Akt signaling and contributed to the protective effects of VEGF on H2O2 induced cell death. Interestingly, SDF-1α also promoted production of VEGF in cultured cardiac myocytes and LY294002 reversed the up-regulation of VEGF induced by SDF-1α.</p><p><b>CONCLUSION</b>VEGF has direct protective effects on cardiomyocytes; a crosstalk between VEGF and SDF-1α through PI3K-Akt serves a survival role in cardiomyocytes in vitro.</p>

Nitric oxide induces heat shock protein 72 production and delayed protection against myocardial ischemia in rabbits via activating protein kinase C / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Nitric oxide (NO) is a biologically active molecule which has been reported to protect the heart against ischemia and reperfusion injury in different species. This study aimed to test the hypothesis that nitric oxide may induce the expression of heat shock protein 72 (HSP72) which may protect the heart against ischemia.</p><p><b>METHODS</b>Rabbits were given intravenous saline or S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, or Zaprinast, an inhibitor of cyclic guanosine monophosphate (GMP)-phosphodiesterase, which may increase myocardial cyclic GMP content. Twenty-four hours later, the rabbits were either sampled to measure HSP72, or induced with a 30-minute coronary occlusion followed by a 120-minute reperfusion, and then the infarct size was measured. Meanwhile, chelerythrine (CHE, an inhibitor of protein kinase C) was given intravenously 5 minutes before SNAP injection and the effect on HSP72 expression and infarct size was determined.</p><p><b>RESULTS</b>Twenty-four hours after pretreatment, immunoblotting showed HSP72 expression increased in the SNAP group compared with control groups, and this was blocked by CHE. Myocardial infarct size in the SNAP group was smaller than that of the control group ((32.4 +/- 5.8)% vs (51.1 +/- 4.7)%, P < 0.05). Pretreated with CHE abolished the infarct size-limiting effect of SNAP ((46.0 +/- 5.1)%). Pretreatment with Zaprinast neither induced HSP72 expression nor reduced infarct size ((55.4 +/- 5.4)%).</p><p><b>CONCLUSION</b>NO induced HSP72 expression and a delayed protection to the heart via the activities of protein kinase C by a cyclic GMP-independent pathway.</p>

Toxintyping of Clostridium perfringens Strains by Colony Multiplex PCR / 微生物学通报

Four primers against the genes encoding(cpa,cpb,etx,and iA) four major toxins(?,?,?,?) of Cl. perfringens were designed and the colony multiplex PCR of identification and genotyping of Cl. perfringens strains were developed. Cl. perfringens reference strains stored in china institute of veterinary drug control including A,B,C,D and E genotyping were genotyped using the colony muitiplex PCR assay. The expected sequences were obtained successfully by the colony multiplex PCR assay. But the sequences were not obtained from Cl. novyi,Cl. septicum and Cl. tetani. The expected sequences were obtained from Cl. perfringens individual colony diluted to 100 times with 0.85% saline solution.13 Cl. perfringens strains isolated from diferent animals were genotyped using the colony multiplex PCR assay,and the results were comparaed with the results of toxins neutranization test in mice. The two assays showed good accordance. These results showed that the development of the colony multiplex PCR is very important for early and fast identification and genotyping of Cl. perfringens in china.