Objective To detect Mycobacterium tuberculosis (MTB)and its resistance against rifampin (RIF ) by applying Xpert MTB/RIF assay in the clinical samples from patients with extrapulmonary tuberculosis,and to discuss the value of this assay in extrapulmonary tuberculosis. Methods Totally 300 clinical samples from the patients who were highly suspected with extrapulmonary tuberculosis and hospitalized in Department of Tuberculosis Section,Shanghai Public Health Clinical Center of Fudan University from May 2014 to May 2015 were collected.Smear and fluorescence staining microscopy,MGIT 960 BACTEC liquid culture,Xpert MTB/RIF assay were applied to detect MTB in these samples.Meanwhile,all the patients received the peripheral blood T-cell spot of tuberculosis test (T-SPOT.TB).The sensitivity and specificity of Xpert MTB/RIF assay for MTB and its resistance against rifampin (RIF)in extrapulmonary tuberculosis samples were evaluated.The measurement data of two independent samples were analyzed by using t test and enumeration data were analyzed by usingχ2 test.Results Totally 282 out of 300 clinical samples from patients with extrapulmonary tuberculosis were included in this study,and 62.7% were male with average age of (32.1 ±24.6 )years and 37.3%were female with average age of (37.8 ±21 .0)years.When MGIT 960 BACTEC liquid culture results were considered as standard diagnosis,the sensitivity,specificity,positive predictive value and negative predictive value of Xpert MTB/RIF assay were 53.8% (95 %CI :37.4%—69.6%),100.0% (95 %CI :86.3%—100.0%),100.0% (95 %CI :80.8%—100.0%)and 63.3% (95 %CI :48.3%—76.2%), respectively.The sensitivity of Xpert MTB/RIF assay in smear-positive/culture-positive samples was 92.3% (95 %CI :62.1 %— 99.6%),while that in smear-negative/culture-positive samples was only 34.6% (95 %CI :17.9%— 55 .6%).Xpert MTB/RIF assay had advantage for detecting MTB in fine needle aspirates,pus,stool and urine,but not in serous effusion and cerebrospinal fluid.However,the sensitivities of MGIT BACTEC 960 liquid culture,fluorescence smear and T-SPOT.TB were 25 .8%, 21 .8% and 70.2%,respectively,and the specificities were 95 .5 %,90.3% and 51 .6%,respectively. The diagnostic value of Xpert MTB/RIF assay was not significantly different from those of MGIT BACTEC 960 liquid culture and fluorescence smear (χ2 =0.61 and 3.45 ,respectively;both P >0.05 ), while it was significantly different from T-SPOT.TB (χ2 =50.58,P <0.05).The sensitivity of T-SPOT. TB was significantly superior to Xpert MTB/RIF assay,but the specificity was relatively low.The sensitivity and specificity of Xpert MTB/RIF assay in RIF resistance detection were 71 .4% (95 %CI :30.3%—94.9%)and 100.0% (95 %CI :86.7%—100.0%),respectively.Conclusions The sensitivity and specificity of Xpert MTB/RIF assay in detecting MTB in fine needle aspirates,pus,stool and urine are relatively high,but not in serous effusion and cerebrospinal fluid.However,the pros of rapid detection and the ability of detecting RIF resistance are useful for the diagnose of extrapulmonary tuberculosis in clinical settings.

Objective To evaluate the value of enzyme-linked immunospot assay (TB ELISPOT) combined with serum latex agglutination test (LA) for diagnosis of pulmonary tuberculosis plus pulmonary cryptococcosis.Methods Serum and biopsy specimens of 76 patients, who were suspected of pulmonary tuberculosis and/or pulmonary cryptococcosis based on clinical and imaging features, were collected from March 2006 to September 2008 in Shanghai Public Health Clinical Center. TB ELISPOT assay, LA and histopathological examination were performed in all the patients. Results Histopathological and pathogenic examination confirmed pulmonary cryptococcosis in 15 cases and pulmonary tuberculosis in 22 cases, pulmonary tuberculosis plus pulmonary cryptococcosis in 8 cases. The sensitivity and specificity of TB ELISPOT were 91% and 94.4%. The sensitivity and specificity of LA were both 100%. TB ELISPOT assay and LA test were both positive in the 8 cases of pulmonary tuberculosis plus pulmonary cryptococcosis.Conclusions The value of enzyme-linked immunospot assay combined with serum latex agglutination test is high for diagnosis of pulmonary tuberculosis plus pulmonary cryptococcosis.

AIM:To study the regulation of endoplasmic reticulum stress(ERS)using the glucose regulated protein 78(GRP78)/CCAAT/enhancer binding protein homologous protein(CHOP)pathway and explore the related mech-anism of Wenyang-Shengji ointment in facilitating the repair of diabetic refractory wounds.METHODS:To establish a rat model of diabetic refractory wound repair,Sprague-Dawley(SD)rats were fed a high-fat diet and intraperitoneally in-jected with streptozotocin.Subsequently,full-thickness skin defects were induced in the dorsal region of the rats.The ex-periment included 4 groups:normal,model(diabetic refractory wounds),Wenyang-Shengji ointment,and Beifuxin(re-combinant bovine basic fibroblast growth factor gel)groups.The normal and model groups were treated with normal saline after disinfection.In the Wenyang-Shengji ointment and Beifuxin groups,the wounds were topically treated with the re-spective ointments once daily.After 14 d of treatment,wound healing was assessed and quantified using the wound healing rate.Hematoxylin-eosin(HE)staining was employed to examine the micromorphology of the wound tissue.Western blot analysis was performed to measure GRP78,CHOP and caspase-12 levels in the wound tissue.Immunohistochemical analy-sis was used to detect the expression and distribution patterns of GRP78,CHOP and caspase-12 in the wounds.Transmis-sion electron microscopy was used to observe reticulum numbers and swelling.Enzyme-linked immunosorbent assay was used to determine interleukin-1β(IL-1β)level as a pro-inflammatory factor within the wound.RESULTS:Indexes of each group were assessed 14 d after the corresponding intervention.Compared with normal group,the rats in model group exhibited a significant decrease in the wound healing rate(P＜0.01),accompanied by increased inflammatory exudation and poor granulation tissue growth.Additionally,there were increases in the expression levels of GRP78,CHOP and cas-pase-12 proteins(P＜0.01),as well as a significant elevation in the content of inflammatory factor IL-1β(P＜0.01).In contrast,compared with model group,treatment with Wenyang-Shengji ointment resulted in a significant improvement in wound healing rate(P＜0.01),reduction in inflammatory exudation,and enhanced granulation tissue growth(P＜0.01).Furthermore,there was a notable decrease in the protein expression of GRP78/CHOP/caspase-12 within the wound tissue following treatment with Wenyang-Shengji ointment(P＜0.01).The levels of inflammatory factor IL-1β also showed a sig-nificant decrease(P＜0.01).CONCLUSION:Wenyang-Shengji promotes the healing of diabetic refractory wounds,which may be associated with the downregulation of the GRP78/CHOP pathway,inhibition of excessive ERS,and reduc-tion in the level of wound cell apoptosis.

ObjectiveTo investigate the possible mechanism of Wenyang Shengji Ointment （温阳生肌膏， WSO） in the treatment of diabetic wounds with yin syndrome. MethodsA total of 24 SD rats were randomly divided into a group （n=6） and modeling group （n=18）. The modeling group rats were fed with high-fat diet for 14 days and then were injected intraperitoneally with streptozotocin to induce diabetic model. After steroid injection， full-thickness skin defects were created on the back of the rats to establish a diabetic wound with yin syndrome model. The normal group was fed with regular diet， and full-thickness skin defects were created surgically on the back of the rats. The 18 successfully modeled rats were further divided into three groups， the model group， the WSO group， and the Beifuxin （Recombinant Bovine Basic Fibroblast Growth Factor Gel， BX） group， 6 rats in each group. The WSO group was given the ointment to the wound， the Beifuxin group was givne BX gel， and the normal group and model group was disinfected and treated with saline. All groups had their dressings changed once daily for 14 days. Wound healing was recorded on days 0， 3， 7， and 14， and the wound healing rate was calculated on day 3， 7， and 14. On day 14 after treatment， HE staining was performed to observe the pathological morphology of the wound tissue. Western Blot was used to detect the relative protein levels of hypoxia-inducible factor 1 alpha （HIF-1α） and vascular endothelial growth factor （VEGF）. Immunofluorescence was used to measure the fluorescence intensity of HIF-1α in the wound tissue， and ELISA was used to detect the methylglyoxal （MGO） content in the wound tissue. ResultsCompared with the normal group， the model group showed poor wound healing on day 3， 7， and 14， with a low wound healing rate （P＜0.01）. HE staining showed scab coverage on the wound， with inflammatory cell infiltration and disorganized collagen arrangement. The relative protein levels of VEGF were significantly reduced， while the relative protein levels of HIF-1α and the MGO content significantly increased （P＜0.01）， and the fluorescence intensity of HIF-1α was enhanced. Compared to the model group， the WSO group and Beifuxin group showed better wound healing on day 3， 7， and 14， with an increased wound healing rate （P＜0.01）. The wound tissue showed clear and complete epithelial structure， reduced inflammatory cells， mature granulation tissue， and organized collagen arrangement. MGO content was significantly reduced （P＜0.01）. The relative protein levels of HIF-1α and VEGF both significantly increased in the WSO group， while only VEGF increased in the Beifuxin group （P＜0.05 or P＜0.01）. Compared with the Beifuxin group， the WSO group had a thicker epidermal layer， prominent collagen formation， significantly increased HIF-1α fluorescence expression， reduced MGO content in the wound tissue， and higher relative protein levels of HIF-1α （P＜0.05）. ConclusionWSO can reduce the accumulation of MGO in diabetic wound tissue with yin syndrome and activate the HIF-1α/VEGF pathway， which could be one of the mechanisms for promoting wound healing.

Objective To explore the mechanism of Wenyang Shengji Ointment(温阳生肌膏,WYSJO)in the treatment of diabetic wounds from the perspective of network pharmacology,and to veri-fy it by animal experiments. Methods The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and related literature were used to screen active compounds in WYSJO and their corresponding targets.GeneCards,Online Mendelian Inheritance in Man(OMIM),DrugBank,PharmGkb,and Therapeutic Target Database(TTD)databases were employed to identify the targets associated with diabetic wounds.Cytoscape 3.9.0 was used to map the ac-tive ingredients in WYSJO,which was the diabetic wound target network.Search Tool for the Retrieval of Interaction Gene/Proteins(STRING)platform was utilized to construct protein-protein interaction(PPI)network.Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)enrichment analyses were performed to identify signaling pathways be-tween WYSJO and diabetic wounds.AutoDock 1.5.6 was used for molecular docking of core components in WYSJO to their targets.Eighteen rats were randomly divided into control,model,and WYSJO groups(n=6).The model and WYSJO groups were used to prepare the model of refractory wounds in diabetes rats.The wound healing was observed on day 0,5,9,and 14 after treatment,and the wound tissue morphology was observed by hematoxylin-eosin(HE)staining.The expression levels of core genes were detected by quantitative real-time polymerase chain reaction(qPCR). Results A total of 76 active compounds in WYSJO,206 WYSJO drug targets,3 797 diabetic wound targets,and 167 diabetic wound associated WYSJO targets were screened out through network pharmacology.With the use of WYSJO-diabetic wound target network,core targets of seven active compounds encompassing quercetin,daidzein,kaempferol,rhamnetin,rham-nocitrin,strictosamide,and diisobutyl phthalate(DIBP)in WYSJO were found.GO enrich-ment analysis showed that the treatment of diabetes wounds with WYSJO may involve lipopolysaccharide,bacteria-derived molecules,metal ions,foreign stimuli,chemical stress,nutrient level,hypoxia,and oxidative stress in the biological processes.KEGG enrichment analysis showed that the treatment of diabetes wounds with WYSJO may involve advanced glycation end products(AGE-RAGE),p53,interleukin(IL)-17,tumor necrosis factor(TNF),hypoxia inducible factor-1(HIF-1),apoptosis,lipid,atherosclerosis,etc.The results of animal experiments showed that WYSJO could significantly accelerate the healing process of diabetic wounds(P<0.05),alleviate inflammatory response,promote the growth of granulation tis-sues,and down-regulate the expression levels of eight core genes[histone crotonyltrans-ferase p300(EP300),protoc gene-oncogene c-Jun(JUN),myelocytomatosis(MYC),hypoxia inducible factor 1A(HIF1A),mitogen-activated protein kinase 14(MAPK14),specificity pro-tein 1(SP1),tumor protein p53(TP53),and estrogen receptor 1(ESR1)]predicted by the net-work pharmacology(P<0.05). Conclusion The mechanism of WYSJO in treating diabetes wounds may be closely related to AGE-RAGE,p53,HIF-1,and other pathways.This study can provide new ideas for the phar-macological research of WYSJO,and provide a basis for its further transformation and appli-cation.