Retinal ischemia-reperfusion injury and pretreatment with Lycium barbarum glycopeptide

AIM:To investigate the antioxidant protective effect of Lycium barbarum glycopeptide(LbGP)pretreatment on retinal ischemia-reperfusion(I/R)injury(RIRI)in rats.METHODS:RIRI was induced in Sprague Dawley rats through anterior chamber perfusion,and pretreatment involved administering LbGP via gavage for 7d.After 24h of reperfusion,serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),and creatinine(CREA)levels,retinal structure,expression of Caspase-3 and Caspase-8,superoxide dismutase(SOD)activity,and malondialdehyde(MDA)in the retina were measured.RESULTS:The pretreatment with LbGP effectively protected the retina and retinal tissue from edema and inflammation in the ganglion cell layer(GCL)and nerve fiber layer(NFL)of rats subjected to RIRI,as shown by light microscopy and optical coherence tomography(OCT).Serum AST was higher in the model group than in the blank group(P=0.042),but no difference was found in ALT,AST,and CREA across the LbGP groups and model group.Caspase-3 expression was higher in the model group than in the blank group(P=0.006),but no difference was found among LbGP groups and the model group.Caspase-8 expression was higher in the model group than in the blank group(P=0.000),and lower in the 400 mg/kg LbGP group than in the model group(P=0.016).SOD activity was lower in the model group than in the blank group(P=0.001),and the decrease was slower in the 400 mg/kg LbGP group than in the model group(P=0.003).MDA content was higher in the model group than in the blank group(P=0.001),and lower in the 400 mg/kg LbGP group than in the model group(P=0.016).The pretreatment with LbGP did not result in any observed liver or renal toxicity in the model.CONCLUSION:LbGP pretreatment exhibits dosedependent anti-inflammatory,and antioxidative effects by reducing Caspase-8 expression,preventing declines of SOD activity,and decreasing MDA content in the RIRI rat model.

Lycium barbarum glycopeptide(wolfberry extract)slows N-methyl-N-nitrosourea-induced degradation of photoreceptors

Photoreceptor cell degeneration leads to blindness, for which there is currently no effective treatment. Our previous studies have shown that Lycium barbarum(L. barbarum) polysaccharide(LBP) protects degenerated photoreceptors in rd1, a transgenic mouse model of retinitis pigmentosa. L. barbarum glycopeptide(Lb GP) is an immunoreactive glycoprotein extracted from LBP. In this study, we investigated the potential protective effect of Lb GP on a chemically induced photoreceptor-degenerative mouse model. Wild-type mice received the following: oral administration of Lb GP as a protective pre-treatment on days 1–7;intraperitoneal administration of 40 mg/kg N-methylN-nitrosourea to induce photoreceptor injury on day 7;and continuation of orally administered Lb GP on days 8–14. Treatment with Lb GP increased photoreceptor survival and improved the structure of photoreceptors, retinal photoresponse, and visual behaviors of mice with photoreceptor degeneration. Lb GP was also found to partially inhibit the activation of microglia in N-methyl-N-nitrosourea-injured retinas and significantly decreased the expression of two pro-inflammatory cytokines. In conclusion, Lb GP effectively slowed the rate of photoreceptor degeneration in N-methyl-N-nitrosourea-injured mice, possibly through an anti-inflammatory mechanism, and has potential as a candidate drug for the clinical treatment of photoreceptor degeneration.

Reversal of Apoptotic Resistance by Lycium barbarum Glycopeptide 3 in Aged T Cells

Objective To study whether Lycium barbarurn glycopeplide 3 （LBGP3） affects T cell apoptosis in aged mice. Methods LBGP3 was purified with DEAE cellulose and Sephadex columns. Apoptotic ＂sub-G1 peak＂ was detected by flow cytometry and DNA ladder was resolved by agarose gel electrophoresis. Levels of IFN-7 and IL-10 were measured with specific kits and mRNA expression was detected by RT-PCR. Apoptosis-related proteins of FLIP, FasL, and Bcl-2 were determined by Western blotting. Results LBGP3 was purified from Fructus Lycii water extracts and identified as a 41 kD glycopeptide. Treatment with 200 p.g/mL LBGP3 increased the apoptotic rate of T cells from aged mice and showed a similar DNA ladder pattern to that in young T cells. The reversal of apoptotic resistance was involved in down-regulating the expression of Bcl-2 and FLIP, and up-regulating the expression of FasL. Conclusion Lycium barbarum glycopeptide 3 reverses apoptotic resistance of aged T cells by modulating the expression of apoptosis-related molecules.

Experimental Study on the Enhancement of Murine Splenic Lymphocyte Proliferation by Lycium Barbarum Glycopeptide

In order to investigate the immunoactivity of Lycium Barbarum glycopeptide (LBG), the routinely prepared murine splenic lymphocyte suspension was separately added into the samples with different concentrations (500, 100, 10, 1 μg/ml) of LBG as LBG groups. Blank control group in the absence of Lycium Barbarum glycopeptide or ConA and positive control group in the presence of 0.5 ml ConA but in the absence of LBG were created. 0.5 ml LBG samples with different concentrations in combination with 0.5 ml ConA (10 μg/ml) into each well to observe the synergic effects of LBG and ConA as LBG+ConA groups. After incubation for 72 h at 37 ℃, the samples were analyzed by CFSE-labeled cells combined with flow cytometry, and MTT. Flow cytometry revealed that both LBG could enhance the murine splenic lymphocyte proliferative reaction. Combined use of LBG and ConA had synergic effects. MTT demonstrated that sample A could obviously promote the murine splenic lymphocyte proliferative reaction as compared with control group (P<0.01), while sample B could also enhance the lymphocyte proliferation at a high dose. In combination with ConA, sample A had synergic effects at high dose, while sample B showed obviously synergic effects (P<0.05). It was concluded that both samples (A and B) had strong immunocompetence.

Effects of laminin glycopeptides on metastasis-related behaviors of cancer cells

Our previous reports have shown that lamininglycopeptides (LN-GPs), the total glycopeptides prepared from laminin (LN), can prevent the experimental lung metastasis and liver metastasis of mouse cancer cells. In order to explore the anti-metastatic mechanism of LNGPs, we studied the effects of LN-GPs on metastasisrelated behaviors of cancer cells in vitro. LN-GPs did not affect cell survival. However, LN-GPs inhibited cell attachment and spreading Of 5180 cells on LN- and Matrigelsubstrate in dose-dependent and time-dependent manners. Moreover, inhibition of cen attachment and spreading on Matrigel substrates were much greater on Matrigel substrate than on LN substrate. In the presence of LN-GPs, 5180 cells on LN substrate changed from a flattened polygonal shape to a round one, the migration of 5180 cells on LN substrate decreased, and the number of a highly invasive human pulmonary giant caxcinoma PG cells invading Matrigel filter in a Boyden chamber was reduced. LN-GPs thus have multiple inhibitory effects on cancer motastasisrelated behaviors.

Generation of induced secretome from adipose-derived stem cells specialized for disease-specific treatment:An experimental mouse model

BACKGROUND Recently,the exclusive use of mesenchymal stem cell(MSC)-secreted molecules,named as the secretome,have been evaluated for overcoming the limitations of cell-based therapy while maintaining its advantages.AIM To improve cell-free therapy by adding disease-specificity through stimulation of MSCs using disease-causing materials.METHODS We collected the secretory materials(named as inducers)released from AML12 hepatocytes that had been pretreated with thioacetamide(TAA)and generated the TAA-induced secretome(TAA-isecretome)after stimulating adipose-derived stem cells with the inducers.The TAA-isecretome was intravenously administered to mice with TAA-induced hepatic failure and those with partial hepatectomy.RESULTS TAA-isecretome infusion showed higher therapeutic potential in terms of(1)restoring disorganized hepatic tissue to normal tissue;(2)inhibiting proinflammatory cytokines(interleukin-6 and tumor necrosis factor-α);and(3)reducing abnormally elevated liver enzymes(aspartate aminotransferase and alanine aminotransferase)compared to the naïve secretome infusion in mice with TAA-induced hepatic failure.However,the TAA-isecretome showed inferior therapeutic potential for restoring hepatic function in partially hepatectomized mice.Proteomic analysis of TAA-isecretome identified that antioxidant processes were the most predominant enriched biological networks of the proteins exclusively identified in the TAA-isecretome.In addition,peroxiredoxin-1,a potent antioxidant protein,was found to be one of representative components of TAA-isecretome and played a central role in the protection of TAA-induced hepatic injury.CONCLUSION Appropriate stimulation of adipose-derived stem cells with TAA led to the production of a secretome enriched with proteins,especially peroxiredoxin-1,with higher antioxidant activity.Our results suggest that appropriate stimulation of MSCs with pathogenic agents can lead to the production of a secretome specialized for protecting against the pathogen.This approach is expected to open a new way of developing various specific therapeutics based on the high plasticity and responsiveness of MSCs.

Structural Analysis of an Oligosaccharide and Glycopeptide Mixture from Panax Ginseng Root with Inhibition SHP-1 Function

A mixture of oligosaccharide and glycopeptide was isolated from the aqueous extract of Panax ginseng roots. The mixture inhibits protein tyrosinc phosphatase（SHP-1） function, implying it enhances immune activity. The peak molecular mass of the oligosaccharide portion is 1800 calculated via GPC software after separation by HPLC. And the structure of the oligosaccharide portion is the backbone of （1→3）- and （1→4）-linked arabinopyranoside, and （1→4）- and （1→6）-linked glucopyranoside, with non-reducing terminals of arabinopyranoside and glucopyranoside. The peak molecular mass of glycopeptide portion is 1900 calculated via GPC software after separation by HPLC. The structure of glycopeptide portion is the backbone of （1→3）- and （1→4）-linked arabinopyranoside, and （1→3,6）-linked glucopyranoside, with non-reducing terminals of galactopyranose and glucopyranoside. The peptide composition is Glu. Asp, Hyp, Set, Arg, Gly , Thr, Pro, Ala, Val, lie, Leu and Lys. The oligosaccharide-peptide linkage is formed by Ara and Hyp.

Online Effective Identification of Glycopeptide Using Liquid Chromatography Combined with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS)

The detailed glycan structural analysis of glycoprotein is amenable to glycopeptide enrichment. Here, we develop a simple, effective and economical approach to enrich glycopeptides from proteolytically digested peptide mixtures by chromatographic column packed with graphite carbon and activated charcoal (G/A-column). Glycopeptide from ovalbumin was efficiently enriched by homemade G/A-column using liquid chromatography and the structure of glycopeptide was obtained by tandem mass spectrometry using Fourier transform ion cyclotron resonance mass spectrometry. The results in this study demonstrate that G/A-column can be used to enrich N-glycolpeptides and be benefit for online identification of glycopeptide using LC-MS.

Optimized extraction and purification technology of Gekko sulfated glycopeptide based on its anticancer activities

Objective:To optimize the extraction and purification technologies of Gekko sulfated glycopeptide based on the content of glycopeptide,removal ratio of proteins,and anticancer activities.Methods:Different extraction methods,namely,water extraction,ultrasonic extraction,enzymatic hydrolysis-water extraction,and enzymatic hydrolysis-ultrasonic extraction were considered to determine the best extraction method.Single factor and orthogonal experiments were performed to determine the optimum extracting conditions.Sevage,enzymatic hydrolysis-Sevage,trichloroacetic acid(TCA),TCA-Sevage and enzymatic hydrolysis-TCA methods were tested to determine the best deproteinization method.The glycopeptide content and protein removal ratio were analyzed by the phenol-sulfuric acid and Coomassie Brilliant Blue methods.Results:Gekko sulfated glycopeptide obtained by water extraction could inhibit the proliferation of HepG2 and SKBR3,as well as promote that of lymphocytes.The glycopeptide content was 4.049%in the optimal extracting condition of a triple decoction extraction for 1 hour each time with a material to solvent ratio of 1:15.The enzymatic hydrolysis-TCA method was found to be the optimal deproteinization method,with a protein removal ratio of 50.46%,glycopeptide content of 14.27%,and inhibitory ratio on human HepG2 cells of 49.06%.Conclusion:This extraction and purification technique for Gekko sulfated glycopeptide is reasonable,feasible,and provides a scientific basis for industrial production.

Enzymatic recovery of glycopeptides from different industrial grades edible bird’s nest and its by-products:nutrient,probiotic and antioxidant activities,and physicochemical characteristics

This study was conducted to recover edible bird’s nest(EBN)hydrolysates from different grades of EBN,including the industrial by-products,using enzymatic treatment.The nutrient,physicochemical properties and antioxidant activities of the recovered hydrolysates at different hydrolysis times were evaluated.Results showed that the recovery yield of enzymatic hydrolysis was above 89%for all grades of EBN and the degree of hydrolysis increased over time.Nitrite content(0.321-0.433 mg/L)was below the permissible tolerance level for all samples.Interestingly,the antioxidant activities(DPPH and ABTS scavenging activities and ferric reducing antioxidant powder(FRAP)activity)were significantly higher(P≤0.05)in hydrolysates recovered from EBN by-products(EBNhC and EBNhD)as compared to the high grade EBN hydrolysates(EBNhA and EBNhB).The in-vitro probiotic activity of EBN and its hydrolysates were examined using the probiotic bacterium Lactobacillus plantarum.Evidently,EBN by-products hydrolysate(EBNhD)recorded the highest number of L.plantarum(1.1×1011 CFU/mL),indicating that low grade EBN has the potential as prebiotic material that promotes probiotic activity.This study demonstrated the concept of using EBN by-products hydrolysates for various applications,such as functional ingredients with enhanced bioactivities,to improve its economic value.

The effects of Gekko sulfated glycopeptide and basic fibroblast growth factor on human fibrosarcoma HT1080 cells

Background:Fibrosarcoma is a malignant soft tissue tumor of mesenchymal origin.Gekko sulfated glycopeptide(GSPP),an anticancer drug in traditional Chinese medicine,could inhibited the tumor angiogenesis by targeting basic fibroblast growth factor(bFGF).bFGF promoted the proliferation of fibroblasts.Both fibrosarcoma and fibroblasts derived from fibrous connective tissue.This study investigated whether GSPP has the inhibitory effects on human fibrosarcoma HT1080 cells.Materials and methods:The trypan blue exclusion assay was used to determine cell viability and cell numbers.Cells migration was observed by wound-healing and transwell.Results:From the first day to seventh day,HT1080 cells number of GSPP,bFGF,GSPP combined bFGF groups had not change compared with control.HT1080 cells migration distance and the number of migrating cells of GSPP,bFGF,GSPP combined bFGF groups were not significantly reduced.Conclusions:GSPP did not have inhibitory effects on the proliferation and migration of human fibrosarcoma HT1080 cells.Thus further research should be carried out in order to study the mechanism of GSPP and bFGF acting on the tumor stroma.

Biomechanics mechanism of the anti-tumor effects of sulfated polysaccharide and sulfated glycopeptide

There are also a variety of cytokines in the tumor microenvironment,which are involved in the change in the hardness of the tumor,thereby affecting the invasion and metastasis of tumor cells.As traditional Chinese medicines,drugs of softening and dissipating firm knot contains different kinds of sulfated polysaccharide and sulfated glycopeptide.By inhibiting the function of cancer-associated fibroblasts,they reduce interstitial fibrosis,thereby reducing the hardness of the tumor and exerting an anti-tumor effect.

Different Regulation Effects of Gekko Sulfated Glycopeptide in Breast and Liver Cancer Cell Lines

Background:Our previous work demonstrated that Gekko sulfated glycopeptide extracted from the Chinese gecko Shou gong(Gekko swinhonis Guenther)could inhibit tumor growth by regulating basic fibroblast growth factor.However,basic fibroblast growth factor has opposing effects on growth in breast and liver cancers.Direct effects and mechanisms of Gekko sulfated glycopeptide on tumor growth remain unclear and are ripe for further exploration.Methods:Differential regulation by Gekko sulfated glycopeptide and bFGF were studied in established human breast cancer MCF-7 cells and hepatocarcinoma HepG2 cells.Cell proliferation was evaluated with a Trypan blue exclusion assay.Cell cycle phases were measured by flow cytometry.Basic fibroblast growth factor,transforming growth factor-β1,and p21WAF1/CIP1 mRNA and protein expression levels were detected by real-time PCR(mRNA)and ELISA(protein).Changes in phosphorylated extracellular signal-regulated kinase levels were analyzed by Western blot.Results:Data indicated that Gekko sulfated glycopeptide inhibited the proliferation of HepG2 cells(P<0.001)and also blocked basic fibroblast growth factor-stimulated proliferation of these cells(P=0.001).Gekko sulfated glycopeptide was shown to increase transforming growth factor-β1 and p21WAF1/CiP1 expression(P<0.01)and partially compensate for reductions therein induced by basic fibroblast growth factor.Conversely,in MCF-7 cells,Gekko sulfated glycopeptide alone had no observable effect on transforming growth factor-β1 and p21WAF1/CiP1 expression.Gekko sulfated glycopeptide did,however,enhance basic fibroblast growth factor-induced inhibition of cell proliferation and transforming growth factor-β1 and p21WAF1/CiP1 expression in the MCF-7 cells(P=0.001,P<0.01,P<0.01,respectively).Gekko sulfated glycopeptide was shown to suppress basic fibroblast growth factor secretion in both HepG2 and MCF-7 cell lines(P<0.05 and P<0.01,respectively)and inhibited extracellular signal-regulated kinase 1/2 phosphorylation facilitated by basic fibroblast growth factor.Gekko sulfated glycopeptide alone decreased phosphorylated extracellular signal-regulated kinase in HepG2 cells but did not visibly affect phosphorylated extracellular signal-regulated kinase levels in MCF-7 cells.Conclusions:Gekko sulfated glycopeptide,a basic fibroblast growth factor inhibitor,differentially regulates growth in different cancer cell lines,and these differences may be determined by the opposing effects of basic fibroblast growth factor on transforming growth factor-β1 and p21WAF1/CiP1 levels in breast and liver cancer cells.

枸杞糖肽可减轻放射治疗后人牙龈成纤维细胞来源的外泌体导致的成骨抑制

目的研究枸杞糖肽(LbGP)对放射后人牙龈成纤维细胞(HGFs)来源的外泌体对成骨抑制的保护作用。方法将HGFs分为对照组、放射组及枸杞糖肽组,通过RT-qPCR、Western blotting及β-半乳糖苷酶染色检测细胞的凋亡、衰老水平以及α-平滑肌肌动蛋白(α-SMA)的差异表达。提取各组细胞分泌的外泌体,通过电镜观察、粒径分析以及Western blotting鉴定外泌体,并分别将各组牙龈成纤维细胞产生的外泌体作用于破骨前体细胞及骨髓间充质干细胞(BMSCs)中,通过Trap染色、茜素红染色、ALP染色、RT-qPCR以及Western blotting检测细胞的破骨及成骨情况。结果衰老细胞在放射组较对照组增多,而在枸杞糖肽组较放射组减少;与对照组相比,放射组Bcl-2/Bax降低(P<0.0001),α-SMA表达水平增加(P<0.05);与放射组相比,枸杞糖肽组Bcl-2/Bax增多(P<0.05),α-SMA表达水平降低(P<0.01);与对照组相比,放射组破骨细胞增多,枸杞糖肽组破骨细胞减少;钙结节、碱性磷酸酶表达水平在放射组低于对照组,在枸杞糖肽组高于放射组;成骨相关基因(BMP2、ALP、RUNX2)及成骨相关蛋白(ALP、RUNX2)的表达水平在枸杞糖肽组高于放射组(P均<0.01)。结论枸杞糖肽可通过作用于放射后的牙龈成纤维细胞的外泌体,有效抑制破骨、促进成骨和预防放射性颌骨骨髓炎的发展,这可能为放射性颌骨骨髓炎的治疗提供一种新策略。

荞麦糖肽脂质体的制备及其稳定性和缓释研究

制备一种荞麦糖肽(buckwheat glycopeptide,BG)缓释脂质体,提高荞麦糖肽的生物利用度。本研究首先对制备所得的BG进行稳定性分析,随后利用反向蒸发法制备荞麦糖肽脂质体(buckwheat glycopeptide liposomes,BGL),经响应面实验对包埋工艺进行优化,并探究其稳定性、体外缓释特性、α-葡萄糖苷酶(α-glucosidase,AGase)抑制活性和抗氧化活性。结果表明:温度(20~80℃)对BG的活性影响较小,碱性条件下BG易失活。从5种磷脂中筛选出蛋黄卵磷脂制备BGL,响应面优化工艺条件为磷脂与胆固醇质量比为4.2∶1.0,脂药比为7.5∶1.0,水相体积为1 mL,此条件下,BGL包埋率为72.64%。BGL对高温(100℃)环境不耐受,在短时间(1 h)内具有广谱pH(2~10)稳定性,反复冻融5次对其渗漏率无显著影响(P>0.05)。BGL在体外模拟胃、肠体系中表现出良好的缓释特性,24 h累计释放速率分别为(84.50±2.30)%和(60.17±1.42)%。

TGase催化的壳寡糖糖基化修饰玉米肽的抗氧化活性

为进一步提高玉米肽的抗氧化活性,以玉米醇溶蛋白源活性肽为原料,利用谷氨酰胺转氨酶(TGase)催化壳寡糖(分子质量1500 Da)与玉米肽共价结合制备玉米糖肽,研究壳寡糖的糖基化修饰对玉米肽抗氧化活性的影响,并对玉米糖肽进行体外模拟胃肠消化,表征玉米糖肽在体内发挥抗氧化活性的潜力。结果表明:TGase催化的壳寡糖共价结合使玉米肽的肽含量和体外消化率显著提高;在质量浓度为2.0 mg/mL时,TGase催化壳寡糖糖基化修饰使玉米肽的DPPH自由基、羟自由基、超氧阴离子自由基清除率,Fe^(2+)螯合率和还原力提高,体外消化使玉米糖肽的DPPH自由基、羟自由基清除率和还原力提高,而Fe^(2+)螯合率和超氧阴离子自由基清除率降低。综上,TGase途径的壳寡糖糖基化反应使玉米肽的抗氧化活性显著提高,且体外消化可以促进玉米糖肽抗氧化活性的释放(Fe^(2+)螯合能力和超氧阴离子自由基清除能力除外)。

度拉糖肽联合二甲双胍对肥胖型2型糖尿病患者机体代谢、体脂成分及血清脂肪因子的影响

目的 探究度拉糖肽联合二甲双胍在肥胖型2型糖尿病(T2DM)患者治疗中的临床疗效。方法 将2021年1月至2023年1月在上海市嘉定区安亭医院接受治疗的200例符合本课题筛选条件的肥胖型T2DM患者,随机分为利拉鲁肽组(n=100)和度拉糖肽组(n=100)。利拉鲁肽组给予利拉鲁肽联合二甲双胍治疗,度拉糖肽组给予度拉糖肽联合二甲双胍治疗,2组均治疗3个月。比较治疗前及治疗3个月机体代谢指标[空腹血糖(FBG)、餐后2 h血糖(2 h PBG)、血红蛋白(HbA1c)、总胆固醇(TC)、甘油三酯(TG)]、体脂成分(体脂肪率、体重指数、四肢皮下脂肪率、内脏脂肪指数)及血清脂肪因子[脂联素(adiponectin)、神经肽Q(NPQ)、白脂素(asprosin)、鸢尾素(irisin)]水平;观察2组临床疗效及不良反应发生情况。结果 治疗3个月,2组FBG、2 h PBG、HbAlc、TC、TG、体脂肪率、体重指数、四肢皮下脂肪率、内脏脂肪指数、asprosin均较治疗前降低,且度拉糖肽组低于利拉鲁肽组(P<0.05)。治疗3个月后,2组血清adiponectin、NPQ、irisin水平均较治疗前升高,且度拉糖肽组升高幅度大于利拉鲁肽组(P<0.05)。度拉糖肽组有效率98.00%高于利拉鲁肽组91.00%(P<0.05)。2组不良反应发生率(11.00%、14.00%)相比,无统计学差异(P>0.05)。结论 度拉糖肽联合二甲双胍可改善肥胖型T2DM患者机体代谢状态,调节机体体脂成分及血清脂肪因子,临床疗效显著,安全性高。

Combining lymph node ratio to develop prognostic models for postoperative gastric neuroendocrine neoplasm patients

BACKGROUND Lymph node ratio(LNR)was demonstrated to play a crucial role in the prognosis of many tumors.However,research concerning the prognostic value of LNR in postoperative gastric neuroendocrine neoplasm(NEN)patients was limited.AIM To explore the prognostic value of LNR in postoperative gastric NEN patients and to combine LNR to develop prognostic models.METHODS A total of 286 patients from the Surveillance,Epidemiology,and End Results database were divided into the training set and validation set at a ratio of 8:2.92 patients from the First Affiliated Hospital of Soochow University in China were designated as a test set.Cox regression analysis was used to explore the relationship between LNR and disease-specific survival(DSS)of gastric NEN patients.Random survival forest(RSF)algorithm and Cox proportional hazards(CoxPH)analysis were applied to develop models to predict DSS respectively,and compared with the 8th edition American Joint Committee on Cancer(AJCC)tumornode-metastasis(TNM)staging.RESULTS Multivariate analyses indicated that LNR was an independent prognostic factor for postoperative gastric NEN patients and a higher LNR was accompanied by a higher risk of death.The RSF model exhibited the best performance in predicting DSS,with the C-index in the test set being 0.769[95%confidence interval(CI):0.691-0.846]outperforming the CoxPH model(0.744,95%CI:0.665-0.822)and the 8th edition AJCC TNM staging(0.723,95%CI:0.613-0.833).The calibration curves and decision curve analysis(DCA)demonstrated the RSF model had good calibration and clinical benefits.Furthermore,the RSF model could perform risk stratification and individual prognosis prediction effectively.CONCLUSION A higher LNR indicated a lower DSS in postoperative gastric NEN patients.The RSF model outperformed the CoxPH model and the 8th edition AJCC TNM staging in the test set,showing potential in clinical practice.

枸杞糖肽对角膜碱烧伤小鼠角膜炎症反应及愈合的作用及其机制研究

目的探讨枸杞糖肽(LbGp)在角膜碱烧伤损伤愈合中的作用及其机制。方法根据治疗方式不同将45只健康、SPF级、6~8周、雄性C57BL/6J小鼠随机分为正常对照组(N组)、损伤对照组(PBS组)、LbGp治疗组(LbGp组),每组15只。PBS组和LbGp组小鼠右眼复制碱烧伤模型,采用点眼联合结膜下注射的方式分别给予PBS溶液或LbGp溶液(10 mg/mL)治疗。治疗后第3天采用裂隙灯显微镜及HE染色观察各组小鼠角膜上皮修复情况和角膜组织结构;实时荧光定量聚合酶链反应、Western blotting及免疫组织化学染色技术检测各组小鼠角膜组织NOD样受体热蛋白结构域相关蛋白3(NLRP3)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、髓过氧化物酶(MPO)的表达及核转录因子κB(NF-κB)的磷酸化水平。治疗后的第14天采用裂隙灯显微镜对角膜混浊程度进行评分,HE染色观察角膜组织结构。结果治疗后第3天LbGp组小鼠角膜上皮愈合速度快于PBS组(P<0.05)。N组、PBS组及LbGp组的NF-κB-p65、NLRP3、IL-1β及IL-6基因和蛋白相对表达量比较,差异均有统计学意义(P<0.05),PBS组均高于N组(P<0.05),LbGp组均低于PBS组(P<0.05)。LbGp组角膜基质中MPO染色阳性的中性粒细胞的浸润较PBS组减轻。治疗后第14天LbGp组小鼠角膜混浊评分低于PBS组(P<0.05)。结论LbGp治疗可以抑制小鼠角膜碱烧伤后NF-κB的激活及NLRP3、IL-1β的表达,从而减轻过度的炎症反应,促进小鼠角膜上皮再生及角膜结构恢复,有助于恢复角膜的透明性和完整性。

酶法制备玉米七肽糖基化产物及其对乙醇脱氢酶激活活性的影响研究

目的对玉米七肽(Ser-Ser-Asn-Cys-Gln-Pro-Phe,SSNCQPF)进行糖基化制备和分离纯化,并对其进行乙醇脱氢酶(alcohol dehydrogenase,ADH)激活活性验证。方法通过谷氨酰胺转氨酶(transglutaminase,TGase)介导,以玉米七肽作为酰基供体,D-氨基葡萄糖(D-glucosamine,GlcN)为酰基受体合成玉米糖肽的糖基化反应,并分离纯化玉米糖肽单体[SSNCQ(GlcN)PF]。使用超高效液相色谱-静电场轨道阱质谱仪(ultra performance liquid chromatography-quadrupole exactive orbitrap mass spectrometry,UPLC-QE Orbitrap MS)进行面积归一化法测定糖肽纯度,采用核磁共振波谱仪(nuclear magnetic resonance,NMR)测定糖肽的氢谱(1 H NMR)和碳谱(^(13)C NMR),并结合二级质谱进行结构定性分析。最后用酶标仪(microplate reader spectrometry,MRS)对比测定玉米七肽及其糖肽的ADH激活活性,以验证玉米七肽糖基化修饰对其ADH激活活性的影响。结果玉米糖肽经过分离、纯化和冻干后,其纯度达到99.11%,并且通过二级质谱、核磁氢谱和碳谱的分析,确定氨基葡萄糖连接在玉米七肽谷氨酰胺残基的氨基上。在摩尔浓度为2.5 mmol/L时,糖肽的ADH激活率比玉米七肽高9.89%。结论与玉米肽相比,采用TGase酶介导玉米七肽和氨基葡萄糖合成的糖肽,具有更高的ADH激活活性,为玉米肽糖基化在食品工业中的应用提供参考。