AIM: To identify the primary active components and underlying mechanisms of Yijing Decoction(YJD)in treating early diabetic retinopathy(DR)based on liquid chromatography-mass spectrometry and network pharmacology.METHODS: Active components of YJD were characterized through LC-MS. Components with optimal ADME(absorption, distribution, metabolism, excretion)properties were selected as key bioactive candidates. Network pharmacology approaches were employed to predict YJD-DR therapeutic targets. Protein-protein interaction(PPI)networks, gene ontology(GO)enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were subsequently conducted to predict core targets and networks. Critical targets and pathways were experimentally validated through Western blot.RESULTS: Ten core therapeutic targets were identified, including TNF, Alb, EGFR, STAT3, PTGS2, ESR1, PPAR, MMP9, TLR4, and MAPK. YJD was related to cancer-related signaling, fluid shear stress and atherosclerosis, and neurodegenerative diseases, encompassing key biological processes such as inflammatory response regulation, programmed cell death activation, and enhanced cell migration. Furthermore, Western blot analysis confirmed that YJD significantly inhibited high glucose-induced phosphorylation of STAT3(P-STAT3/STAT3)and ERK(P-ERK/ERK)in rat retinal microvascular endothelial cells.CONCLUSION: This study revealed YJD's pharmacodynamical basis and its multi-component, multi-target, and multi-paths pharmacology. YJD exerts therapeutic effects on DR by coordinately regulating critical signaling pathways and alleviating intraocular inflammation, thus preserving retinal vascular endothelial cells, maintaining blood-retinal barrier integrity, and facilitating retinal neurovascular repair.

OBJECTIVE To develop a DNA authenticity identification kit of Gastrodia elata that combined DNA extraction technology with PCR technology, and to evaluate the performance of the kit methodologically. METHODS The ITS2 sequences of Gastrodia elata and its common forgeries, such as amabilis root, dahlia tuber and potato, were found by the National Center for Biotechnology Information(NCBI). DNAMAN was used for multi-sequence alignment, and NCBI-primer-blast was used to design specific primers of Gastrodia elata. Improved DNA extraction method to ensure efficient extraction of authentic Gastrodia elata and its common forgeries genomic DNA, UV spectrophotometry was used to measure the concentration and purity. The PCR reaction system was optimized, the composition and reaction conditions of the kit were determined, and the commercially available gastrodia elata samples were randomly sampled. RESULTS The DNA purity OD260/OD280 values of the samples extracted by the developed kit were (1.87±0.13). The minimum detection limit was 10 ng·μL−1, and the result of repeated detection was the same for 3 times. Repeated freezing and thawing for 5, 10, 15, 20 times had no effect on the detection effect, and it could be stored at −20℃ for 1 year, among 10 commercially available gastrodia elata samples tested, 7 were authentic and 3 were counterfeit. CONCLUSION The DNA authenticity test kit is highly specific, sensitive, reproducible and stable, and the test results are accurate, it is suitable for the rapid identification of asparagus and its common forgeries.

Objective:To establish a method for rapid detection of OXA and par C resistance genes of Acinetobacter baumannii(Ab)by double nucleic acid colloidal gold strip and to develop kit.Methods:DNA of Ab was extracted by heating and boiling method.OXA and par C genes sequences of Ab were selected as target gene fragments based on NCBI.Primers were designed and labeled with 6-FAM,digoxin and biotin,respectively.Drug resistance gene detection reagents were developed,and nucleic acid gold test strips were used for rapid and visual detection.Molecular cloning and sequencing techniques were used to clone positive control samples and evaluate specificity,sensitivity and stability of kit.Results:DNA concentration and purity of Ab extracted by boiling method were good.Homology between cloned and sequenced plasmid DNA and gene sequence in GenBank database was 100%,respectively.Speci-ficity of kit was good,with only Ab showing positive results and other bacterial genera showing negative results;DNA concentration of Ab in double nucleic acid colloidal gold test strip decreased to 10-3 ng/μl,a red line still appeared,whose sensitivity was 10 times higher consistent with minimum detection limit of electrophoresis 10-2 ng/μl;test kits were tested at 3rd,6th and 9th months,and showed good stability.Conclusion:Double resistance detection kit established in this study can simultaneously detect OXA and par C resis-tance of Ab,who has advantages of high sensitivity,strong specificity,rapid and simple,and provides a new method for detection of carbapenem and quinolone antibiotic resistance of Ab.

Background: Brucella infection induces brucellosis, a zoonotic disease. The intracellular circulation process and virulence of Brucella mainly depend on its type IV secretion system (T4SS) expressing secretory effectors. Secreted protein BspJ is a nucleomodulin of Brucella that invades the host cell nucleus. BspJ mediates host energy synthesis and apoptosis through interaction with proteins. However, the mechanism of BspJ as it affects the intracellular survival of Brucella remains to be clarified. Objectives: To verify the functions of nucleomodulin BspJ in Brucella's intracellular infection cycles. Methods: Constructed Brucella abortus BspJ gene deletion strain (B. abortus ΔBspJ) and complement strain (B. abortus pBspJ) and studied their roles in the proliferation of Brucella both in vivo and in vitro. Results: BspJ gene deletion reduced the survival and intracellular proliferation of Brucellaat the replicating Brucella-containing vacuoles (rBCV) stage. Compared with the parent strain, the colonization ability of the bacteria in mice was significantly reduced, causing less inflammatory infiltration and pathological damage. We also found that the knockout of BspJ altered the secretion of cytokines (interleukin [IL]-6, IL-1β, IL-10, tumor necrosis factor-α, interferon-γ) in host cells and in mice to affect the intracellular survival of Brucella. Conclusions BspJ is extremely important for the circulatory proliferation of Brucella in the host, and it may be involved in a previously unknown mechanism of Brucella's intracellular survival.

Objective:To explore the clinical efficacy of aspirin plus low molecule heparin for pancreatic thrombosis during simultaneous pancreas and kidney transplantation (SPK).Methods:A total of 129 patients aged 18 years or higher underwent SPK between September 2016 and March 2020.They were divided retrospectively into two groups of aspirin ( n=60) and heparin ( n=69) according to different anticoagulant regimens.The aspirin group received only aspirin 100 mg/d at Day 1 post-operation.The heparin group received subcutaneous injection of enoxaparin 2 000 AxaIU daily for 7 days and followed by aspirin and clopidogrel.Outcomes and complication rates were compared between two groups. Results:All operations were successful without any mortality.In aspirin group, there were 5 cases of pancreatic thrombosis and one patient underwent pancreatectomy.There was no pancreatic thrombosis in heparin group ( P=0.014). There were 8 cases of intestinal anastomotic bleeding in aspirin group and 19 cases in heparin group.Statistically significant inter-group difference existed ( P=0.048). However, no significant inter-group difference existed in delayed recovery or rejection. Conclusions:Heparin anticoagulation can significantly lower the incidence of pancreatic thrombosis after SPK.Despite a higher incidence of intestinal anastomotic bleeding, no serious complication occurs after conservative meaures.

Objective To investigate the safety of young recipients undergoing living donor renal transplantation from elderly relative donors through long-term follow-up of the pathological changes. Methods According to the age of donors, 28 young recipients were divided into the observation group (n=14, elderly donors) and control group (n=14, young and middle-aged donors). The 7-year survival after renal transplantation, the serum creatinine (Scr) levels at various postoperative time points were compared between two groups. The chronic pathological injury scores of renal allograft biopsy at time-zero, postoperative 6-month and 7-year were compared between two groups. The expression levels of renal interstitial fibrosis indicators connective tissue growth factor (CTGF), transforming growth factor (TGF)-β, laminin (LN), fibronectin (FN), cell senescence indicators intercellular connexin (Cx)-43 and mammalian target of rapamycin (mTOR) at postoperative 6-month and 7-year were compared between two groups. Results The 7-year survival rates in the observation and control groups were 78.5% and 92.8% with no statistical significance (P > 0.05). In the observation and control groups, the levels of Scr were 190 and 160 μmol/L at the postoperative 7 d, and 170 and 125 μmol/L at postoperative 1 month. At each postoperative time point, the levels of Scr in the observation group were significantly higher than those in the control group (all P > 0.05). The total chronic pathological injury scores of renal transplant biopsy at time-zero in the observation group was significantly higher than that in the control group (P > 0.05), whereas the total chronic pathological injury scores at postoperative 7-year did not significantly differ between two groups (P > 0.05). Within either group, the total chronic pathological injury scores at postoperative 7-year was remarkably higher than those at time-zero and postoperative 6-month (both P < 0.05). The expression levels of CTGF, TGF-β, LN, FN, mTOR, Cx43 of renal transplant tissue at postoperative 7-year did not significantly differ between two groups (all P > 0.05). Conclusions The long-term follow-up outcomes demonstrate that the pathological changes of young recipients undergoing renal transplantation from elderly donors are similar to those from young and middle-aged donors. It is safe and feasible for young recipients to undergo renal transplantation from elderly donors in the pathological perspective.

Objective To investigate the role of cyclic GMP-AMP synthase (cGAS),a cytosolic DNA sensor,in regulating innate immune responses induced by reverse transcription intermediate of human T cell leukemia virus type 1 (HTLV-1). Methods (1)ssDNA90,the reverse transcription intermediate of HTLV-1,was transfected into HeLa cells to observe changes in the expression pattern of cGAS in transfected-HeLa cells with immunoblot assay. (2) HeLa cells were firstly transfected with cGAS-encoding plasmid and then ssDNA90 24 hours later. Real-time PCR was used to measure the expression of interferon ( IFN)-β, IFN-gamma-inducible protein 10 ( IP-10 ), regulated on activation, normal T cell expressed and secreted (RANTES) and tumor necrosis factor (TNF)-α. Immunoblot assay was performed to measure phosphorylated interferon regulatory factor 3 (IRF3) and p65. (3)cGAS expression was silenced by siRNA in HeLa and phorbol-12-myristate-13-acetate (PMA)-treated THP1 (PMA-THP1) cells and then ssDNA90 was transfect-ed into these cells 24 hours later. Real-time PCR was used to measure the expression of IFN-β,IP-10,RAN-TES and TNF-α. Immunoblot assay was performed to measure phosphorylated IRF3 and p65. Results Ex-pression of cGAS was increased in HeLa cells after ssDNA90 transfection. Compared with control cells, cGAS-transfected HeLa cells showed increased expression of IFN-β, IP-10, RANTES and TNF-α and en-hanced phosphorylation of IRF3 and p65 after ssDNA90 transfection. Compared with control cells,both HeLa and PMA-THP1 cells with silenced expression of cGAS showed impaired production of IFN-β,IP-10,RAN-TES and TNF-α after ssDNA90 transfection. Moreover,ssDNA90-induced phosphorylation of IRF3 and p65 were decreased after cGAS gene-knockdown. Conclusion cGAS might promote HTLV-1 RTI ssDNA90-in-duced innate immune responses.

Objective To investigate the function and the possible mechanism of cyclic GMP-AMP synthase (cGAS), a DNA sensor, in HeLa cells during human T cell leukemia virus type 1 (HTLV-1) in-fection.Methods HeLa cells were co-cultured with MT2 cells (HTLV-1-positive T cells) and then detec-ted by immunoblot assay to analyze the changes in the expression of cGAS .A hemagglutinin ( HA)-tagged cGAS plasmid was constructed and transfected into HeLa cells .Twenty-four hours after transfection , these cells were co-cultured with MT2 cells for another 24 hours.Immunoblot assay was used to detect the expres-sion of HTLV-1 proteins Tax and p19.Real-time PCR was performed to measure the expression of HTLV-1 Tax, p19, Env, HBZ and px at mRNA level .Immunoblot assay was also used to analyze the phosphorylation of interferon regulatory factor 3 (IRF3) and p65.Expression of interferon (IFN)-β, IFN-gamma-inducible protein 10 ( IP-10 ) , RANTES ( regulated on activation , normal T cell expressed and secreted ) and tumor necrosis factor (TNF)-αwas detected by real-time PCR assay.Results Expression of cGAS was enhanced in HeLa cells after co-cultured with MT2 cells.Compared with control cells , the HeLa cells that were trans-fected with cGAS plasmid showed lower levels of Tax and p 19 proteins, suppressed expression of HTLV-1 Tax, p19, Env, HBZ and px at mRNA level , enhanced phosphorylation of IRF 3 and p65, and higher levels of IFN-β, IP-10, RANTES and TNF-αafter co-cultured with MT2 cells.Conclusion cGAS might promote the innate immune response and inhibit HTLV-1 replication in HTLV-1-infected HeLa cells .

Objective:To evaluate the effect of preoperative panel reactive antibody(PRA)levels on long-term survival after kidney transplantation. Methods:Data on 1 162 patients underwent first kidney transplantation performed between January 2001 and June 2014 were included in our center. According to the preoperative PRA levels,the patients were divided into negative group( PRA≤10%) and positive group( PRA>10%) ,which were retrospectively analyzed. Results: The 1-,5-,10-year patient survival rates of the negative group calculated by Kaplan-Meier were 96. 8%,89. 4%,78. 6%,respectively,while the positive group were 93. 5%,81. 6%, 65. 4%. The 1-,5-,10 -year death-censored graft survival rates of the negative group were 95. 9%,84. 8%,63. 1%,respectively,while the positive group were 92. 3%,74. 1%,51. 9%. The log-rank test revealed that there was significant difference between the patient and graft survival curves (χ2 =9. 623/11. 019, P=0. 002/0. 001 ) . Cox multivariate analysis found that preoperative PRA levels were independent risk factors for reducing the patient or graft survival rates(P<0. 001). Logistic multivariate regression analysis confirmed the significant association between preoperative PRA levels and the risk of acute rejection ( OR=8. 25,95% CI=2. 86-5. 72, P<0. 001). The 5-,10-year creatinine values were significantly lower in the negative group compared to the positive group(all P<0. 05), while there was no difference in the 1-year. In addition, Logistic multivariate regression analysis confirmed the significant association between preoperative PRA levels and the production of donor specific antibody(DSA)(OR=6. 89,95% CI=4. 52-9. 17,P<0. 05). Conclusion: The detection of preoperative PRA is an important indictor predicting the sensitivity status of the recipients. The preoperative PRA positive recipients need careful monitoring and diagnosis of acute rejection and DSA after kidney transplantation.

Objective To study the pathological changes of renal grafts from elderly donor in young recipients and to investigate the safety of kidney transplantation from elderly donors.Methods Fourteen elderly kidney donors (with the age >55 years old)and fourteen young recipients (with the age <30 years old)underwent living related donor renal transplantation at the Department of Transplantation of the Second Affiliated Hospital of Guangzhou Medical University from January 2008 to December 2008 were enrolled as the object of study.Every elderly donor kidney was performed time-zero biopsy and every young recipient was performed routine renal graft biopsy 6 months after transplantation.The pathological and structural changes of kidney tissues after renal transplantation from elderly donors were observed.Results The histopathological changes occurred at 6 months after transplantation from elderly kidney donors to young recipients.Compared with those before transplantation,the severity of renal arteriolar lesion and the index of renal arteriolar sclerosis alleviated after transplantation (P <0.05 ), and the ratio of glomerulosclerosis did not change after transplantation (P >0.05 ).The expression of fibronectin (FN)decreased after transplantation,but the difference had no statistical significance (P >0.05 ).The expression of laminin (LN ) decreased after transplantation (P <0.05).Conclusions The histopathological structure of renal graft from elderly donors in young recipients has improved.