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Objective:To understand the molecular transmission characteristics of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome(AIDS) patients in the mountainous area of southwest Zhejiang Province(Lishui city).Methods:A total of 147 blood samples were collected from newly-diagnosed HIV-1 infected who received no antiviral therapy, and pol gene was amplified, followed by sequencing. MEGA6.0 software was used to construct phylogenetic tree and determine gene subtypes. HIVDB online was used to analyze drug resistance mutation, then the pairwise genetic distance(GD) was calculated and the opitimal threshold of GD was selected, finally the molecular transmission network was constructed by Cytoscape3.7.0 software. Chi-square or Fisher′s exact probability method was used for statistical analysis. Results:A total of 134 sequences were obtained successfully, and nine subtypes were detected. The dominant subtypes were CRF08_BC (34.33%, 46/134), CRF01_AE (29.85%, 40/134) and CRF07_BC (23.88%, 32/134). It also found that age, registered residence, education level and transmission route had significant differences in distribution of subtypes ( P<0.05). Nineteen drug resistance individuals were found, and the total drug resistance rate was 14.18% (19/134). The HIV-1 molecular transmission network was plotted based on 1.2% GD threshold. A total of 15 transmission clusters (cluster size ranging from 2 to 29) were found. The network access rate was 49.25% (66/134), mainly including male (75.76%, 50/66), heterosexual (81.82%, 54/66) and patientsrinfected with CRF08_ BC (50.00%, 33/66). A transmission cluster including two cases of female sex workers and seven cases of drug resistance was identified, in which the average age of the patients was 57.21 years old and the average degree value was 22.7, and the cases were mainly infected through heterosexual contact (96.55%, 28/29). The highest homology of the sequences in the cluster was in Yunnan. Conclusions:The HIV-1 subtypes were diverse in the mountainous area of southwest Zhejiang Province(Lishui city). Drug resistant transmission had reached a moderate epidemic level. There were molecular transmission clusters with the aggregation characteristics of elderly clients in specific regions. It was urgent to formulate and implement precise intervention strategies to curb the spread of HIV.
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Objective:To construct the molecular transmission network of human immunodeficiency virus type 1 (HIV-1) epidemic strains in northern Zhejiang Province (Jiaxing City and Huzhou City) and to explore the HIV-1 transmission characteristics in this region.Methods:A total of 371 newly diagnosed human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) patients in Jiaxing City and Huzhou City in 2017 were included as study subjects, and the blood samples were collected and the basic demographic and epidemiological information were obtained. RNA in plasma was extracted, and the pol region gene sequence was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (PCR) to construct phylogenetic tree for identifying subtypes. The pairwise genetic distances were calculated, and the optimal threshold of genetic distance was selected, and finally the molecular transmission network was constructed. Chi-square test was used for statistical analysis. Results:The pol region gene sequences of 336 samples were successfully obtained, and 11 subtypes were detected, mainly including circulating recombinant form (CRF)07_BC (40.8%, 137/336) and CRF01_AE (31.2%, 105/336). Based on the 1.0% genetic distance threshold, the molecular transmission network of HIV-1 was plotted. A total of 38 transmission clusters (cluster sizes ranging from two to 28) including 119 patients were found, with males predominantly (82.4%, 98/119) and most of the patients aged over 40 (include 40) years old (52.9%, 63/119), mainly infected with CRF07_BC subtype (57.1%, 68/119) and CRF01_AE (24.4%, 29/119). The clustering rate of CRF07_BC (49.6%, 68/137) was significantly higher than that of CRF01_AE (27.6%, 29/105), the difference was statistically significant ( χ2=5.27, P=0.022). Two large clusters C1 (28 cases) and C2 (11 cases) were identified, the majority of which were men who have sex with men (17 cases and seven cases, respectively). High-risk cases generally sought sexual partners in local or nearby cities through mobile phone dating software, of which the infected sequences mostly had high homology with other economic developed regions (Guangdong Province, Beijing City and Hangzhou City, etc.). Conclusions:The HIV-1 subtypes are diverse in Jiaxing City and Huzhou City, mainly CRF07_BC and CRF01_AE. The HIV-1 transmission networks are complex, among which high-risk cases may be the key factor leading to the HIV-1 epidemic in the region. Therefore, it is urgent to deepen the transmission network monitoring and formulate timely precise intervention and prevention strategies.
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Objective To understand the epidemiological characteristics of one large HIV molecular transmission cluster in Jiaxing city,Zhejiang province,2017 in order to select those people under high-risk and providing basis for programs on prevention.Methods During 2017,newly diagnosed HIV/AIDS cases in this city were recruited.Plasma samples were collected from subjects,followed by RNA extraction,RT-PCR and nest-PCR for pol gene amplification,before being sequenced and aligned.Mega 6.0 software was used to construct phylogenetic tree,and Cytoscape 3.6.0 software was used to identify HIV molecular transmission clusters.Cases within the large transmission clusters were investigated,using a field-epidemiology-questionnaire.Data related to socio-demographics and previous sexual behaviors were collected and EpiData 3.0 and SPSS 20.0 software were used.Results In the large transmission cluster with subtype identified as CRF07_BC,in Jiaxing,2017,26 cases of the total 30 cases were investigated.A total of 80.8％ (21/26) could be identified as newly infected within the last two years and 30.8％(8/26) could be identified as newly infected within the last one year,including 22 cases infected locally.Among several infected cases who were at age 45 years or older,they admitted that they had experienced unprotected sexual contacts in local city for long time and having had more than 10 disclosed sexual contacts within the last two years at the local venues.Conclusions This molecular cluster had been formed and scaled up quickly in recent two years,it has played an important role in promoting and scaling up the HIV transmission.Three cases identificed as high risk played an importantrde role in scaling up this cluster.
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Objective To understand the epidemiological characteristics of one large HIV molecular transmission cluster in Jiaxing city,Zhejiang province,2017 in order to select those people under high-risk and providing basis for programs on prevention.Methods During 2017,newly diagnosed HIV/AIDS cases in this city were recruited.Plasma samples were collected from subjects,followed by RNA extraction,RT-PCR and nest-PCR for pol gene amplification,before being sequenced and aligned.Mega 6.0 software was used to construct phylogenetic tree,and Cytoscape 3.6.0 software was used to identify HIV molecular transmission clusters.Cases within the large transmission clusters were investigated,using a field-epidemiology-questionnaire.Data related to socio-demographics and previous sexual behaviors were collected and EpiData 3.0 and SPSS 20.0 software were used.Results In the large transmission cluster with subtype identified as CRF07_BC,in Jiaxing,2017,26 cases of the total 30 cases were investigated.A total of 80.8％ (21/26) could be identified as newly infected within the last two years and 30.8％(8/26) could be identified as newly infected within the last one year,including 22 cases infected locally.Among several infected cases who were at age 45 years or older,they admitted that they had experienced unprotected sexual contacts in local city for long time and having had more than 10 disclosed sexual contacts within the last two years at the local venues.Conclusions This molecular cluster had been formed and scaled up quickly in recent two years,it has played an important role in promoting and scaling up the HIV transmission.Three cases identificed as high risk played an importantrde role in scaling up this cluster.
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Objective@#Using field epidemiological investigation and molecular analysis to construct the molecular transmission network of human immunodeficiency virus/acquired immunodeficiency syndrome cases (HIV/AIDS) newly diagnosed in Huzhou in 2017, Zhejiang Province.@*Methods@#A total of 160 participants were obtained through a web-based system from Chinese Center for Disease Control and Prevention (CCDC) with the features of diagnosed in Huzhou in 2017 who also had been collected samples for the first follow-up. The basic information of demographic characteristics and risk factors was extracted from the website. RNA was extracted from plasma samples of untreated cases, followed by RT-PCR and nest-PCR for pol gene amplification, sequencing. Phylogenetic tree was constructed by MEGA software for HIV gene subtyping. TN93 model was used for calculating the distance between two sequences. Cytoscape software was used for drawing molecular transmission network. And then an epidemiological survey was conducted to cases in the primary cluster.@*Results@#A total of 138 sequenced individuals (86.3%) were acquired from 160 individuals. Among which, 123 (89.1%) were male. The highest proportion of subtype was CRF07_BC (60, 43.5%), followed by CRF01_AE (46, 33.3%), and with four cases of Unique Recombinant Form (URF, CRF01_AE and CRF07_BC) and one case of URF (subtype B and C). A total of 18 molecular clusters included 56 individuals (40.6%) were found in the transmission network under the optimal genetic distance threshold (1.0%). The clustering proportion of CRF07_BC (66.1%, 37 cases) was higher than that of CRF01_AE. There were 9 clusters formed among CRF07_BC, including 37 cases (accounting for 61.7%, 37/60). The primary transmission cluster contained 11 cases, among which 9 cases were transmitted by homosexual sex. The first time of the cases to have homosexual behavior is range from 2010 to 2016, whose media number (P25, P75) of partners was 6 (3.5, 8.5). Most of the cases come from Anhui Province and engaged in garment industry (5 cases), between which there were 8 cases used Blued software to seek for casual partners, 1 case seeking for casual partners in garden.@*Conclusion@#With CRF07_BC and CRF01_AE predominantly circulating, HIV genetic diversity had been noticed in this area. The primary cluster was consisted of high proportion of locally new infections, and a specific population aggregation in limited place existed.
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Objective@#To understand the molecular subtypes and recombinant structure of HIV-1 infection in Zhejiang Province.@*Methods@#According to the method of stratified random sampling method, a total of 302 newly HIV infected people (excluding AIDS patients) were selected as the subjects of the investigation from April to June, 2015, who were all newly HIV positive patients reported in Zhejiang Province and were diagnosed as HIV positive and had not received antiretroviral treatment. Questionnaires were used to collect information such as gender, residency, marital status, route of transmission and case reporting area. At least 8 ml of venous blood was collected from HIV infected people, reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR were used to amplify the pol gene sequence of HIV-1 strains and circulating recombination forms (CRF) and unique recombinant form (URF) strains were screened using phylogenetic tree, and molecular subtype analysis and recombinant analysis were performed.@*Results@#Of the 302 HIV infected people, 18 cases were infected with new CRFs or URFs, among which 15 were male (83.3%), 3 were heterosexual transmission (17.7%); 8 cases (44.4%) were infected with new CRFs, which included CRF55_01B 5(27.8%), CRF67_01B 2 (11.1%), CRF69_01B 1 (5.6%); 10 cases (55.6%) were infected with URFs, in which 6 cases were infected with two groups of recombinant strains composed of CRF01_AE subtype and CRF07_BC subtypes in pol region, 3 cases were infected with three groups of recombinant strains of the two subtypes in pol region, and 1 case could not be accurately determined of the recombinant structure. The results of recombinant source analysis showed that the newly recombinant strains found from Zhejiang province were highly homologous to the strains from Guangdong, Anhui, Hebei, Yunnan, Liaoning province, and the sequence similarity was 94.4%-98.0%.@*Conclusion@#The new recombinant strains of Zhejiang province included CRFs strains and URFs strains with CRF01_AE and CRF07_BC as the main recombinant structure mode, and all of them were introduced from other places and formed a trans-regional chain of HIV transmission.
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Objective To understand the characteristics of distribution on HIV-1 subtypes and the transmission clusters in Yiwu in Zhejiang province.Methods A cross-sectional study of molecular epidemiology was carried out on newly reported H1V/AIDS cases in Yiwu.RNA was extracted from 168 plasma samples,followed by RT-PCR and nest-PCR for pol gene amplification,sequencing,phylogenetic tree construction used for analyzing the subtypes and transmission clusters.Mutations on drug resistance was analyzed by CPR 6.0 online tool.Results Subjects were mainly males (86.3％,145/168),with average age as (39.1 ± 13.4) years old and most of them were migrants (66.7％,112/168).The major routes of transmission included homosexual (51.2％,86/168) and heterosexual (48.8％,82/168) contacts.The rate of success for sequence acquisition was 89.9％ (151/168).The dominant subtypes showed as CRF01_AE (74,49.0％) and CRF07_BC (64,42.4％),followed by CRF08_BC (5,3.3％),CRF55_01B (3,2.0％),each case of subtype B,CRF45_cpx,CRF59_01B,CRF85_BC and URF (B/C).CRF45_cpx and CRF85_BC were discovered the first time in Zhejiang province.Twenty-six transmission clusters involving 65 cases were found,with the total clustered rate as 43.0％ (65/151),in which the CRF01_AE clustered rate appeared as 54.1％ (40/74),higher than that of CRF07_BC (21/64,32.8％).The average size of cluster was 2.5 cases/cluster,with average size of cluster in CRF01_AE patients infected through heterosexual transmission as the largest (3.5 cases/cluster).The prevalence of transmitted drug resistance was 4.6％ (7/151).Seven cases with surveillance drug resistant mutations (SDRM) were found,including 5 cases of M46L (3.3％),and one case of F77L or Y181C.Conclusion HIV genetic diversity and a variety of transmission clusters had been noticed in this study area (Yiwu).Programs on monitoring the subtypes and transmission clusters should be continued and strengthened.
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Objective To understand the distribution of HIV-1 subtype diversity and its transmission characteristics in Zhejiang province. Methods A total of 302 newly diagnosed HIV-1 positive patients were selected through stratified random sampling in Zhejiang in 2015. HIV-1 pol genes were sequenced successfully with reverse transcription PCR/nested PCR and phylogenetic analysis was conducted for 276 patients. Then a molecular epidemiologic study was performed combined with field epidemiological investigation. Results Of 276 sequence samples analyzed, 122 CRF07_BC strains (44.2%), 103 CRF01_AE strains (37.3%), 17 CRF08_BC strains (6.1%), 9 B strains (3.2%), 6 CRF55_01B strains (2.2%), 5 C strains (1.8%), 1 CRF59_01B strain (0.4%), 1 CRF67_01B strain (0.4%), 1 A1 strain (0.4%), and 11 URFs strains (4.0%) were identified. Phylogenetic analysis revealed 16 clusters with only 15.1% (34/225) sequences involved among CRF07_BC and CRF01_AE strains. The clustered cases in MSM were higher than that in populations with other transmission routes. And clusters existed between the populations with different transmission routes. Conclusion The major strains of HIV-1 in Zhejiang are CRF07_BC and CRF01_AE. The HIV subtypes showed more complexity in Zhejiang. It is necessary to strengthen the surveillance for HIV subtypes, carry out classified management and conduct effective prevention and control in the population at high risk.
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Objective To understand the characteristics of distribution on HIV-1 subtypes and the transmission clusters in Yiwu in Zhejiang province.Methods A cross-sectional study of molecular epidemiology was carried out on newly reported H1V/AIDS cases in Yiwu.RNA was extracted from 168 plasma samples,followed by RT-PCR and nest-PCR for pol gene amplification,sequencing,phylogenetic tree construction used for analyzing the subtypes and transmission clusters.Mutations on drug resistance was analyzed by CPR 6.0 online tool.Results Subjects were mainly males (86.3％,145/168),with average age as (39.1 ± 13.4) years old and most of them were migrants (66.7％,112/168).The major routes of transmission included homosexual (51.2％,86/168) and heterosexual (48.8％,82/168) contacts.The rate of success for sequence acquisition was 89.9％ (151/168).The dominant subtypes showed as CRF01_AE (74,49.0％) and CRF07_BC (64,42.4％),followed by CRF08_BC (5,3.3％),CRF55_01B (3,2.0％),each case of subtype B,CRF45_cpx,CRF59_01B,CRF85_BC and URF (B/C).CRF45_cpx and CRF85_BC were discovered the first time in Zhejiang province.Twenty-six transmission clusters involving 65 cases were found,with the total clustered rate as 43.0％ (65/151),in which the CRF01_AE clustered rate appeared as 54.1％ (40/74),higher than that of CRF07_BC (21/64,32.8％).The average size of cluster was 2.5 cases/cluster,with average size of cluster in CRF01_AE patients infected through heterosexual transmission as the largest (3.5 cases/cluster).The prevalence of transmitted drug resistance was 4.6％ (7/151).Seven cases with surveillance drug resistant mutations (SDRM) were found,including 5 cases of M46L (3.3％),and one case of F77L or Y181C.Conclusion HIV genetic diversity and a variety of transmission clusters had been noticed in this study area (Yiwu).Programs on monitoring the subtypes and transmission clusters should be continued and strengthened.
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Objective To understand the distribution of HIV-1 subtype diversity and its transmission characteristics in Zhejiang province. Methods A total of 302 newly diagnosed HIV-1 positive patients were selected through stratified random sampling in Zhejiang in 2015. HIV-1 pol genes were sequenced successfully with reverse transcription PCR/nested PCR and phylogenetic analysis was conducted for 276 patients. Then a molecular epidemiologic study was performed combined with field epidemiological investigation. Results Of 276 sequence samples analyzed, 122 CRF07_BC strains (44.2%), 103 CRF01_AE strains (37.3%), 17 CRF08_BC strains (6.1%), 9 B strains (3.2%), 6 CRF55_01B strains (2.2%), 5 C strains (1.8%), 1 CRF59_01B strain (0.4%), 1 CRF67_01B strain (0.4%), 1 A1 strain (0.4%), and 11 URFs strains (4.0%) were identified. Phylogenetic analysis revealed 16 clusters with only 15.1% (34/225) sequences involved among CRF07_BC and CRF01_AE strains. The clustered cases in MSM were higher than that in populations with other transmission routes. And clusters existed between the populations with different transmission routes. Conclusion The major strains of HIV-1 in Zhejiang are CRF07_BC and CRF01_AE. The HIV subtypes showed more complexity in Zhejiang. It is necessary to strengthen the surveillance for HIV subtypes, carry out classified management and conduct effective prevention and control in the population at high risk.
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<p><b>OBJECTIVE</b>To understand the HIV-1 subtype diversity and transmission characteristics in men having sex with men (MSM) in Zhejiang province.</p><p><b>METHODS</b>A total of 233 newly diagnosed as HIV-1 positive patients in 2011 were screened out by BED capture enzyme immunoassay (BED-CEIA). Among them, 107 eligible subjects were enrolled for further molecular epidemiological study. Viral RNA was extracted from plasma samples and followed by reverse transcription PCR and nested PCR for amplification of pol gene fragments, sequencing, and bioinformatics analysis.</p><p><b>RESULTS</b>There were no statistically significant differences regarding the social demographic distribution between the subjects under study and those recently infected MSM population. The rate of success for sequence acquisition was 94.4% (101/107). The highest proportion of subtype was CRF01_AE (62.4%), followed by CRF07_BC (31.7%) and with three cases of subtype B, one case of CRF55_01B and two cases of unique recombinant form (CRF01_AE/B and CRF01_AE/CRF07_BC). The phylogenetic trees were mainly divided into CRF01_AE cluster 1, cluster 2 and CRF07_BC cluster 3. The strains located in Hangzhou were diffused in the branches of phylogenetic tree. 10 transmission clusters were found, in which 80% involved two or more regions and 90% was associated with patients residing in Hangzhou. Three surveillance drug resistance mutations (M46I, T215S and G190A) were found in three samples (each sample harbored only one resistance mutation). The overall rate of transmitted drug resistance (TDR) was 2.97%.</p><p><b>CONCLUSION</b>The increasing complexity of HIV was noticed in MSM in Zhejiang province. However, the prevalence of TDR was low. Cross-regional HIV transmission in MSM was common, which inferred from the study. Hangzhou might play a central regional role in the intra-provincial spread of HIV, to form an interwoven complex network in the MSM population.</p>
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Humanos , Masculino , China , Epidemiologia , Demografia , Farmacorresistência Viral , Genes pol , Infecções por HIV , Genética , HIV-1 , Classificação , Homossexualidade , Técnicas Imunoenzimáticas , Filogenia , Prevalência , RNA ViralRESUMO
In this study ,we intended to prepare anti‐Toxoplasma gondii aquaporin (TgAQP) peptide antibody which was used to the application in the detection of the aquaporin expression and its subcellular localization of Toxop lasma gondii (T .gondii) ME49 strain .The B cell peptide antigen was designed based on the TgAQP amino acids sequence .After the pep‐tide antigen was conjugated to the KLH ,the fusion antigen was injected into New Zealand rabbits to prepare polyclonal anti‐body ,followed by identification of ELISA ,Western‐blotting and immunofluorescence assays .The ELISA showed that the titer of anti‐TgAQP antibody was about 1∶40 000 .Western blotting revealed the specific affinity of the antigen to polyclonal anti‐body at 29 .9 kDa protein T .gondii .The protein detected by the indirect immunofluorescence assays was distributed in the cy‐toplasm of the parasite .Thus far ,the anti‐TgAQP polyclonal antibody was successfully prepared ,providing a useful tool for further study of biological function and metabolic characteristics of TgAQP .
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Objective To establish a duplex fluorescence PCR for detection of HIV proviral DNA and to evaluate its application for early diagnosis of HIV infection in infants .Methods A duplex fluores-cence PCR system was set up based on TaqMan technology for detection of human ribonuclease P ( RNase P) gene and long terminal repeat ( LTR) region of HIV.A recombinant plasmid containing the targeted gene fragment , pTG19-T, was constructed by TA cloning technique and used as the template for evaluation of sen -sitivity of the assay .Blood samples from 11 healthy individuals and 98 HIV-infected patients were collected and detected to validate the assay specificity .The assay of duplex fluorescence PCR was then carried out to detect 96 infant blood samples collected from several maternal and child health hospitals in Zhejiang province from January 2011 to September 2012 for early diagnosis of HIV infection .The results were compared with those by using the Roche HIV DNA qualitative detection kit .Results The established duplex fluorescence PCR could specifically detect HIV proviral DNA with a specificity of 100%and a detection sensitivity of 100 cps per reaction .The coincidence rate between the established assay and the Roche HIV DNA qualitative de -tection kit was 100%in the detection of 96 blood samples .Conclusion The duplex fluorescence PCR as-say showed advantages of cost-effectiveness , convenience , good specificity and accuracy with high sensitivi-ty.It could be used for early diagnosis of HIV infection in infants and also as a general technical platform for the detection of HIV proviral DNA .
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Objective To investigate the different effects of internal and external biliary drainage on the levels of TNF-α,NO in blood serum and NO,inducible nitric oxide synthase(iNOS) in gastric mucosa with obstructive jaundice and explore the mechanism of gastric mucosa damage and compare the quality of internal and external drainage.Methods To establish the animal model:one hundred male adult Sprague-Dawley rats were randomly assigned to five groups:obstructive jaundice for 7 days group (OJ-7 d group),obstructive jaundice for 14 days (OJ-14 d group),internal biliary drainage group(ID group),external drainage group(ED group)and sham operation group(SH group).The OJ-7 group rats were executed 7 days after the first operation,the rest four groups were executed 7 days after the secondary surgery,leaving serum and gastric mucosa.Tested the levels of INF-α,NO in blood serum and NO,iNOS in gastric mucosa and did pathological biopsy of gastric mucosa.Results The levels of TNF-α,NO in blood serum and NO,iNOS in gastric mucosa significantly increased in OJ-7 group.The levels of TNF-α,NO in blood serum and NO,iNOS in gastric mucosa continued to rise in OJ-14 group,and was significantly higher than OJ-7 group,the gastric mucosa was obviously impaired.The levels of TNF-α,Internal drainage can significantly NO in blood serum and NO,iNOS in gastric mucosa were significantly lower in ID group,gastric mucosal damage significantly reduced.Conclusions After internal drainage,iNOS expression was inhited in gastric mucosal,NO production was significantly reduced,led to protect the gastric mucosa,which provide experimental basis for choice in OJ clinical treatment.