Polypeptide from Moschus Suppresses Lipopolysaccharide-Induced Inflammation by Inhibiting NF-κ B-ROS/NLRP3 Pathway / 中国结合医学杂志

OBJECTIVE@#To examine the anti-inflammatory effects and potential mechanisms of polypeptide from Moschus (PPM) in lipopolysaccharide (LPS)-induced THP-1 macrophages and BALB/c mice.@*METHODS@#The polypeptide was extracted from Moschus and analyzed by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, LPS was used to induce inflammation in THP-1 macrophages and BALB/c mice. In LPS-treated or untreated THP-1 macrophages, cell viability was observed by cell counting kit 8 and lactate dehydrogenase release assays; the proinflammatory cytokines and reactive oxygen species (ROS) were measured by enzyme-linked immunosorbent assay and flow cytometry, respectively; and protein and mRNA levels were measured by Western blot and real-time quantitative polymerase chain reaction (qRT-PCR), respectively. In LPS-induced BALB/c mice, the proinflammatory cytokines were measured, and lung histology and cytokines were observed by hematoxylin and eosin (HE) and immunohistochemical (IHC) staining, respectively.@*RESULTS@#The SDS-PAGE results suggested that the molecular weight of purified PPM was in the range of 10-26 kD. In vitro, PPM reduced the production of interleukin 1β (IL-1β), IL-18, tumor necrosis factor α (TNF-α), IL-6 and ROS in LPS-induced THP-1 macrophages (P<0.01). Western blot analysis demonstrated that PPM inhibited LPS-induced nuclear factor κB (NF-κB) pathway and thioredoxin interacting protein (TXNIP)/nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome pathway by reducing protein expression of phospho-NF-κB p65, phospho-inhibitors of NF-κB (Iκ Bs) kinase α/β (IKKα/β), TXNIP, NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1 (P<0.05 or P<0.01). In addition, qRT-PCR revealed the inhibitory effects of PPM on the mRNA levels of TXNIP, NLRP3, ASC, and caspase-1 (P<0.05 or P<0.01). Furthermore, in LPS-induced BALB/c mice, PPM reduced TNF-α and IL-6 levels in serum (P<0.05 or P<0.01), decreased IL-1β and IL-18 levels in the lungs (P<0.01) and alleviated pathological injury to the lungs.@*CONCLUSION@#PPM could attenuate LPS-induced inflammation by inhibiting the NF-κB-ROS/NLRP3 pathway, and may be a novel potential candidate drug for treating inflammation and inflammation-related diseases.

Tandem expression of the major epitope domains of the Moschus chrysogaster hemorrhagic disease virus VP60 and its protective efficacy to rabbits / 生物工程学报

Moschus chrysogaster (sifanicus) viral hemorrhagic disease (McVHD) is an acute and highly lethal infectious disease caused by Moschus chrysogaster hemorrhagic disease virus (McHDV) whose genome sequence is highly homologous with rabbit hemorrhagic disease virus. To screen the protective antigen of McHDV and set the basis for study of McVHD vaccine, the antigen epitope of major structural protein VP60 of McHDV was analyzed, and the specific primers were designed to obtain three amplified DNA sequences encoding the main antigen epitope of VP60 from McHDV by using RT-PCR. Then the three DNA fragments were sequenced and cloned to prokaryotic expression vector with pET-28a(+) by using overlap extension PCR, and finally the prokaryotic expression plasmid pET-truncated-VP60 was constructed. Subsequently, the pET-truncated-VP60 was transformed into Escherichia coli BL21(DE3), and the recombinant proteins were expressed by IPTG induction. Finally, the expressed protein was purified and applied to immunize that without immunizing with RHD vaccine, then the antiserum titers were evaluated by the hemagglutination inhibition test, and the immune-protective efficacy of the recombinant proteins was observed and analyzed through animal challenge test. The results showed that the multi-epitope DNA fragments of VP60 of McHDV was successfully expressed in the form of inclusion bodies in E. coli, and the relative molecular weight of recombinant proteins is about 45 kDa. After immunized with the recombinant proteins, 100% of New Zealand white rabbits were resistant to attack of McHDV, which indicates efficient immune-protective efficacy of chosen epitope recombinant protein. The study laid a foundation for the development of the new subunit vaccines of McVHD.

Preliminary study on the role of novel LysR family gene kp05372 in Klebsiella pneumoniae of forest musk deer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

LysR-type transcriptional regulators are involved in the regulation of numerous cellular metabolic processes in Klebsiella pneumoniae, leading to severe infection. Earlier, we found a novel LysR family gene, named kp05372, in a strain of K. pneumoniae (designated GPKP) isolated from forest musk deer. To study the function of this gene in relation to the biological characteristics of GPKP, we used the suicide plasmid and conjugative transfer methods to construct deletion mutant strain GPKP-Δkp05372; moreover, we also constructed the GPKP-Δkp05372+ complemented strain. The role of this gene was determined by comparing the following characteristics of three strains: growth curves, biofilm formation, drug resistance, stress resistance, median lethal dose (LD50), organ colonization ability, and the histopathology of GPKP. Real-time polymerase chain reaction (RT-PCR) was used to test the expression level of seven genes upstream of kp05372. There was no significant difference in the growth rates when comparing the three bacterial strains, and no significant difference was recorded at different osmotic pressures, temperatures, salt contents, or hydrogen peroxide concentrations. The GPKP-Δkp05372 mutant formed a weak biofilm, and the other two strains formed medium biofilm. The drug resistance of the GPKP-Δkp05372 mutant toward cephalothin, cotrimoxazole, and polymyxin B was changed. The acid tolerance of the deletion strain was strongerthan that of the othertwo strains. The LD50 values of the wild-type and complemented strains were 174-fold and 77-fold higher than that of the GPKP-Δkp05372 mutant, respectively. The colonization ability of the GPKP-Δkp05372 mutant in the heart, liver, spleen, kidney, and intestine was the weakest. The three strains caused different histopathological changes in the liver and lungs. In the GPKP-Δkp05372 mutant, the relative expression levels of kp05374 and kp05379 were increased to 1.32-fold and 1.42-fold, respectively, while the level of kp05378 was decreased by 42%. Overall, the deletion of kp05372 gene leads to changes in the following: drug resistance and acid tolerance; decreases in virulence, biofilm formation, and colonization ability of GPKP; and regulation of the upstream region of adjacent genes.

Preliminary study on the role of novel LysR family gene kp05372 in Klebsiella pneumoniae of forest musk deer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

LysR-type transcriptional regulators are involved in the regulation of numerous cellular metabolic processes in Klebsiella pneumoniae, leading to severe infection. Earlier, we found a novel LysR family gene, named kp05372, in a strain of K. pneumoniae (designated GPKP) isolated from forest musk deer. To study the function of this gene in relation to the biological characteristics of GPKP, we used the suicide plasmid and conjugative transfer methods to construct deletion mutant strain GPKP-Δkp05372; moreover, we also constructed the GPKP-Δkp05372+ complemented strain. The role of this gene was determined by comparing the following characteristics of three strains: growth curves, biofilm formation, drug resistance, stress resistance, median lethal dose (LD50), organ colonization ability, and the histopathology of GPKP. Real-time polymerase chain reaction (RT-PCR) was used to test the expression level of seven genes upstream of kp05372. There was no significant difference in the growth rates when comparing the three bacterial strains, and no significant difference was recorded at different osmotic pressures, temperatures, salt contents, or hydrogen peroxide concentrations. The GPKP-Δkp05372 mutant formed a weak biofilm, and the other two strains formed medium biofilm. The drug resistance of the GPKP-Δkp05372 mutant toward cephalothin, cotrimoxazole, and polymyxin B was changed. The acid tolerance of the deletion strain was stronger than that of the other two strains. The LD50 values of the wild-type and complemented strains were 174-fold and 77-fold higher than that of the GPKP-Δkp05372 mutant, respectively. The colonization ability of the GPKP-Δkp05372 mutant in the heart, liver, spleen, kidney, and intestine was the weakest. The three strains caused different histopathological changes in the liver and lungs. In the GPKP-Δkp05372 mutant, the relative expression levels of kp05374 and kp05379 were increased to 1.32-fold and 1.42-fold, respectively, while the level of kp05378 was decreased by 42%. Overall, the deletion of kp05372 gene leads to changes in the following: drug resistance and acid tolerance; decreases in virulence, biofilm formation, and colonization ability of GPKP; and regulation of the upstream region of adjacent genes.

Microbial diversity of musk across its maturation process in forest musk deer / 中国中药杂志

Musk,with unique and intense perfume,was a kind of deep brown precious medicinal material in traditional Chinese medicine. However,the immature musk in musk pot was white and stench. Given the fact that bacterial diversity generated odorous metabolites in animal hosts,in this study,musk samples at three different mature stages,including MJ( the end of June),MA( the end of August) and MO( the end of October) were harvested from three male forest musk deer,and then next-generation sequencing was used to intensively survey the bacterial communities in musk harvested at different mature stages. RESULTS: indicated that the average OTUs per sample at the end of June,August and October were 47 116. 00 ± 1 567. 24( SE),52 009. 00 ± 8 958. 75( SE) and50 004. 67±4 135. 57( SE),respectively. Feature of the musk 16 S rRNA gene showed a total of 418 genera belonging to 52 phyla were observed in all samples. The main microbiota was bacteria,which accounted for 98. 82%,99. 95% and 99. 58% in MJ,MA and MO,respectively. At phylum level,Firmicutes was the most abundant bacterial of MA( 32. 75%) and MO( 39. 19%). While,the major bacterial in MJ was Proteobacteria( 49. 14%). PICRUSt analysis revealed the functions of bacterial in MJ were mainly involved in secretion,while bacterial functions of MA and MO were mainly involved in amino acid or other substance metabolism,which was in accord with the musk secretion physiological process of forest musk deer. This is the first study involved in the bacterial diversity in musk of forest musk deer across the maturation process,while may provide a new insight into the musk generation mechanism.

Developmental toxicity of muscone on zebrafish embryos / 中国药理学与毒理学杂志

OBJECTIVE To investigate the develop mental toxicity of muscone to embryos. METHODS With zebrafish embryos as a model,The 3 h post fertilization (hpf)embryos were exposed to muscone at 5,10,20,40,80 and 160 μmol·L -1 culture solutions for 96 h and inspected daily with mi-croscopy for larval morphology.The drug solution was replaced every 24 h.Spontaneous move ments were checked at 24 hpf.Heart rate at 48 hpf,hatching rate,e mbryo deformity rate and mortality rate were evaluated.The expression of sepn1 was determined with real-ti me quantitative PCR technique at 96 hpf.RESULTS The 24 hpf spontaneous move ments showed no significant difference.At 48 hpf, spine curvature,pericardial ede ma,yolk sac ede ma,and abnormal swi mming were observed.In addition, the 48 hpf heart beats(10 s)was decreased fro m 26.5 ±1 .0 to 18.0 ±1 .9(P <0.01 ).At 48 hpf , hatching rate of 5 ~40 μmol·L -1 decreased(P <0.05),while of 160 μmol·L -1 increased (P <0.05) co mpared with muscone 0 μmol·L -1 .Muscone had little effect on hatching rate at other ti me points;Mal-formation rate and mortality rate at higher concentrations were up to 100%.The sepn1 gene expression at 96 hpf in the exposure groups decreased co mpared with that of control group(P <0.01 ).CONCLU-SION Muscone had toxic effects on the develop ment of zebrafish embryos,including spine curvature, abnormal swi mming,and pericardial ede ma.These effects may be related to the inhibition of sepn1 gene expression by muscone.

Study on Extraction of Moschus from Shehuang-Babuji / 中国药房

OBJECTIVE:To optimize the extraction technique of moschus and formulate the preparation procedure for Shehuang-babuji.METHODS:Concentration of alcohol,amount of alcohol,frequencies of extraction,extraction time were taken as variable factors affecting the extraction rate of moschus,the L 9 (3 3 )orthogonal test was adopted.RESULTS:The opti?mal extraction technique was heated recirculation for3times with2hours each time in95%alcohol12times the amount of herb medicinal material;The quality evaluation of various indices for the product met the standards.CONCLUSION:The extraction technique of moschus is feasible and reliable.

Effect of Moschus Combined with Borneol on Brain Water Content and Blood-Brain Barrier Permeability in Rat Model of Cerebral Focal Ischemia with Reperfusion / 广州中医药大学学报

【Objective】To observe the effect of Moschus combined with Borneol on brain water content and blood-brain barrier(BBB)permeability in rat model of cerebral focal ischemia with reperfusion.【Methods】Thirty SD rats were randomized into 6 groups: sham-operation group,model group,Moschus(1 mg?kg-1?d-1)group,Borneol(3 mg?kg-1?d-1)group,Moschus with Borneol group,nimodipine(12 mg?kg-1?d-1)group.The rat model of cerebral focal ischemia with reperfusion was established with method of putting nylon monofilament in the internal carotid artery.The brain water content was evaluated by detecting the wet weight and dry weight of brain.The detection of cerebral extra-vascular Evans blue(EB)content was used to observe the changes of BBB permeability.【Results】After cerebral ischemia for 2 hours and reperfusion for 24 hours,water content and EB content in the model group increased significantly compared with the pseudo-operation group(P

Therapeutic Effect of Resuscitation-inducing Aromatic Herbs for Acute Ischemic Stroke / 广州中医药大学学报

To investigate the effect of treatment based on syndrome differentiation combined with resuscitation inducing aromatic herbs for acute ischemic stroke (AIS).Forty four cases of AIS were randomly allocated to two groups. Twenty cases of Group A were treated with treatment based on syndrome differentiation combined with Tongqiao capsule(each capsule containing Moschus 0 04?g and Borneolum 0 1?g)and twenty four of Group B with treatment based on syndrome differentiation. Therapeutic effect were observed in the second week after medication. The total effective rate was 95% in Group A and 91 6% in Group B, the difference being not significant. However, the difference of markedly effective rate (80 0% in Group A and 54 2% in Group B) was significant (P

Protective Effect of Xing Nao Jing on Cultured Cortical Neurons in Rats / 广州中医药大学学报

[Objective] To study the protective effect of Xing Nao Jing (XNJ) on cultured cortical neurons in rats. [Methods] Primary cultured cortical neurons were applied to observe the effect of XNJ in counteracting the excitotoxicity ofglutamic acid. [Results] XNJ decreased the release of intracellular lactic dehydrogenase induced by glutamic acid and reduced the histological changes of cultured cortical neurons. [Conclusion] XNJ can counteract the excitotoxicity of glutamic acid and protect cultured cortical neurons in rats.

COMPARISON STUDY OF EFFECTS OF CALCULUS AND MOSCHUS TOTAL EXTRACT AND HYDROCORTISONE ON MYOCARDIAL FUNCTION IN DOGS FOLLOWING CORONARY ARTERY OCCLUSION / 中国药理学通报

This study was aimed to compare the anti-inflammatory calculus bovis and moschus ( C + M) with the typical steroid hydrocortisone (H) in their effects on regional ischemic myocardium. 22 anesthetized dogs were studied by the mercnry-in-Silastic length gauge and divided into Saline (S) ( n= 7 ) , C + M ( n= 8 ) and H ( n=7 ) groups. Within 3 h after occlusion of left anterior descending coronary artery (LAD), the phasic segmental length ( ASL) was reduced significantly in the II groups, indicating that regional myocardial function was improved.The amplitude of VSL in the (C + M) group, however, was not obviously changed at 1,2 and 3 h after occlusion LAD. This was identical with that of the Saline group. These results show that though C + M and H were both anti-inflammatory agents in peripheral tissues as mentioned in previous reports, C + M had no steroid effect in ischemic myocardium.