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Nano-LC-MS/MS was used to analyze trypsin digested deer-horn gelatin( DCG) and deer-hide gelatin( DHG) samples.The glycopeptides in DCG and DHG were quantified by Label-free quantitative( LFQ) peptidomics,on the basis of which the glycopeptides with significant difference in DCG and DHG were determined. As a result,5 736 peptides were identified from DCG samples,including 213 galactosyl-hydroxylysine containing peptides( Gal-Hyl-peptides) and 102 glucosyl-galactosyl-hydroxylysine containing peptides( Glc-Gal-Hyl-peptides),while 6 836 peptides were identified from DHG samples,among which there were 250 Gal-Hyl-peptides and 98 Glc-Gal-Hyl-peptides. With over 3-fold peak area difference and highly significant intergroup difference( P < 0. 01) as the screening criteria,444 differential peptides were determined in DCG and DHG,including 16 Gal-Hyl-peptides and 5 Glc-Gal-Hyl-peptides. Then XIC peak shapes,standard deviation of peak area,and fold change were applied for further screening and 5 glycopeptides with significant differences in DCG and DHG were confirmed,which could serve as potential biomarkers for distinguishing DCG and DHG. The present study provided ideas and strategies for the in-depth investigation on the discrimination of DCG and DHG and is of good theoretical significance and application value for the further research on chemical constituents and quality control of gelatin derived Chinese medicinals.
Subject(s)
Animals , Chromatography, Liquid , Deer , Gelatin , Glycopeptides , Tandem Mass SpectrometryABSTRACT
Our previous studies have shown that puerarin, an active component of the traditional Chinese medicine-Pueraria Lobata, can improve glycometabolism in high-fat diet (HFD) mice with diabetes by activating the glucagon-like peptide-1 receptor (GLP-1R) pathway. This study intends to further evaluate the effect of puerarin on depressive symptoms in HFD mice. Long-term HFD induces type 2 diabetes and depressive-like symptoms in mice. Animal welfare and experimental procedures follow the regulations of the Animal Ethics Committee of the Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Traditional Chinese Medicine (approval No. AEWC-025). The experiment was divided into: control group, model group, model/puerarin (150 mg·kg-1·day-1) group, and model/fluoxetine (15 mg·kg-1·day-1) group. The oral glucose tolerance test (OGTT) and behavioral experimental analysis were performed after 6 weeks of continuous administration. Afterwards, enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin-1β (IL-1β), interleukin-6 (IL-6), 5-hydroxytryptamine (5-HT), and corticosterone (CORT) in serum of mice for each group. Western blot assays were used to detect the level of activation and expression of proteins related to neuroplasticity and depressive disorder in the hippocampus. Moreover, HT-22 cell line was used to investigate the protective effect of puerarin on cell morphology and survival. The results show that puerarin can effectively maintain the survival of HT22 in an environment with high glucose and corticosterone. Meantime, the glycemic regulation of diabetic mice was improved after treatment of puerarin, the depressive symptoms were alleviated, the 5-HT increased, and the corticosterone, IL-1β, and IL-6 decreased in the serum. The up-regulation of related proteins in GLP-1R/Wnt/mTOR (mammalian target of rapamycin) signaling in hippocampus suggests that its effect on ameliorating depression in diabetic mice may be related to the activation of GLP-1R/Wnt/mTOR signaling pathway. This study shows that puerarin can significantly ameliorate the depressive symptoms of HFD induced diabetic mice which might be achieved through activating the GLP-1R/Wnt/mTOR signaling pathway and improving hippocampal neuroplasticity.
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Nano-LC MS/MS was used to analyze trypsin digested deer-hide gelatin(DHG) samples, hydroxylation and O-glycosylation on lysine sites of DHG were comprehensive identified by using PEAKS Studio software. The sites, sorts and amounts of hydroxylation and O-glycosylation on Type Ⅰ collagen α1 chain(COL1 A1) and α2 chain(COL1 A2) of DHG were revealed. As a result, 5 284 peptides were identified from DHG samples, which were mainly from COL1 A1 and COL1 A2. Among these peptides, there were 449 peptides with hydroxylysine, 442 with galactosyl-hydroxylysine, 449 with glucosyl-galactosyl-hydroxylysine. The major modified sites of hydroxylation and O-glycosylation in DHG were shown as follow: α1-9 N and α2-5 N in N-telopeptides, α1-87, α1-174, α1-930, α2-87, α2-174, α2-933 in triple helix domain, and α1-16 C in C-telopeptides. These hydroxylation and O-glycosylation were correlated with the formation and stability of collagen molecules and collagen fibrils. It is feasible for the collagens and peptides dissolving from deer skin collagen fibrils under high temperature and pressure decocting, high temperature and pressure also might destroy inter-molecular covalent cross-linking and help those glycol-peptides formations. The present study provided ideas and strategies for the in-depth investigation on DHG chemical constituents, and showed good theoretical significance and application value.
Subject(s)
Animals , Deer/metabolism , Gelatin , Glycosylation , Hydroxylation , Lysine/metabolism , Protein Processing, Post-Translational , Tandem Mass SpectrometryABSTRACT
Objective: As an important food therapy product with traditional Chinese medicine (TCM) applications, donkey-hide gelatin (Asini Corii Colla, ACC) has been used for thousands of years. However, till now few effective strategy had been proposed to distinguish ACC from other animal hide gelatins, especially closely related horse- and mule-hide gelatins, which was an embarrassment of ACC quality control. Methods: Combined mass spectrometry and bioinformatic methods have been applied to identify and verify two ACC-specific peptides (Pep-1 and Pep-2) capable of distinguishing ACC from other closely related animal gelatins with high selectivity. Results: It confirmed that these two peptides could be not only used for distinguishing ACC from highly homologous horse-hide and mule-hide gelatins as well as other animal hide gelatins. Conclusion: The present study provides a simple method for species-specific peptides discovery, which can be used for assessing the quality of animal gelatin products, and ensure they are authenticable and traceable.
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Objective@#To explore the diagnostic value of combined detection of serum human epididymis protein 4 (HE4) and carbohydrate antigen 125 (CA125) for ovarian epithelial cancer.@*Methods@#Patients who underwent surgery for the adnexal tumor at Shanxi Provincial Cancer Hospital from January 2012 to December 2017 were enrolled. According to the postoperative pathological results, these patients were divided into the ovarian epithelial cancer group (494 cases) and benign ovarian disease group (462 cases). The serum expressions of HE4 and CA125 in the two groups were detected by enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay. The diagnostic value of detection of HE4 and CA125 alone or in combination for ovarian epithelial cancer was analyzed.@*Results@#The median levels (P 25 - P 75) of serum CA125 and HE4 in ovarian epithelial cancer group were 273.34 U/ml (39.34 U/ml, 709.74 U/ml) and 199.08 pmol/L (75.81 pmol/L, 449.20 pmol/L), which were higher than those in ovarian benign disease group [16.30 U/ml (6.30 U/ml, 53.60 U/ml) and 39.54 pmol/L (29.57 pmol/L, 53.80 pmol/L)] (both P < 0.05). There was a positive correlation between serum CA125 and HE4 levels in ovarian epithelial cancer group (r = 0.481, P < 0.01). Serum CA125 and HE4 levels in patients with stage Ⅲ and Ⅳ were higher than those in patients with stage Ⅰ and Ⅱ (both P < 0.05), and serum CA125 and HE4 levels in patients with poor differentiation were higher than those in patients with moderate differentiation (P < 0.05). Compared with CA125, the specificity and positive predictive value of serum HE4 for the diagnosis of ovarian epithelial cancer were higher (both P < 0.01). Compared with HE4 alone, the sensitivity of CA125 combined with HE4 increased (P = 0.004) and the specificity decreased (P = 0.044). When both CA125 and HE4 were positive for positive results, compared with HE4 alone, the specificity and positive predictive value increased (both P < 0.01), but the sensitivity decreased (both P < 0.01). In patients with CA125+ HE4-, the sensitivity and specificity decreased, while in CA125- HE4+ patients, the specificity was elevated, but the difference was not statistically significant (P = 0.892). When one of CA125 and HE4 was positive for positive results, the sensitivity and negative predictive value increased (both P < 0.01), but the specificity and positive predictive value decreased (both P < 0.01). The area under the curve of serum CA125 and HE4 combined detection was 0.911, indicating its clinical diagnostic value was better than that of the two alone.@*Conclusions@#In the diagnosis of epithelial ovarian cancer, the specificity of serum HE4 is higher than that of CA125, but the sensitivity is lower than that of CA125. The combined detection of HE4 and CA125 is more conducive to improve the diagnostic accuracy of ovarian epithelial cancer.
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Objective To explore the diagnostic value of combined detection of serum human epididymis protein 4 (HE4) and carbohydrate antigen 125 (CA125) for ovarian epithelial cancer. Methods Patients who underwent surgery for the adnexal tumor at Shanxi Provincial Cancer Hospital from January 2012 to December 2017 were enrolled. According to the postoperative pathological results, these patients were divided into the ovarian epithelial cancer group (494 cases) and benign ovarian disease group (462 cases). The serum expressions of HE4 and CA125 in the two groups were detected by enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay. The diagnostic value of detection of HE4 and CA125 alone or in combination for ovarian epithelial cancer was analyzed. Results The median levels (P 25-P 75) of serum CA125 and HE4 in ovarian epithelial cancer group were 273.34 U/ml (39.34 U/ml, 709.74 U/ml) and 199.08 pmol/L (75.81 pmol/L, 449.20 pmol/L), which were higher than those in ovarian benign disease group [16.30 U/ml (6.30 U/ml, 53.60 U/ml) and 39.54 pmol/L (29.57 pmol/L, 53.80 pmol/L)] (both P< 0.05). There was a positive correlation between serum CA125 and HE4 levels in ovarian epithelial cancer group (r=0.481, P<0.01). Serum CA125 and HE4 levels in patients with stage Ⅲ and Ⅳ were higher than those in patients with stageⅠand Ⅱ (both P<0.05), and serum CA125 and HE4 levels in patients with poor differentiation were higher than those in patients with moderate differentiation (P< 0.05). Compared with CA125, the specificity and positive predictive value of serum HE4 for the diagnosis of ovarian epithelial cancer were higher (both P< 0.01). Compared with HE4 alone, the sensitivity of CA125 combined with HE4 increased (P= 0.004) and the specificity decreased (P= 0.044). When both CA125 and HE4 were positive for positive results, compared with HE4 alone, the specificity and positive predictive value increased (both P< 0.01), but the sensitivity decreased (both P< 0.01). In patients with CA125+ HE4-, the sensitivity and specificity decreased, while in CA125-HE4+patients, the specificity was elevated, but the difference was not statistically significant (P= 0.892). When one of CA125 and HE4 was positive for positive results, the sensitivity and negative predictive value increased (both P<0.01), but the specificity and positive predictive value decreased (both P<0.01). The area under the curve of serum CA125 and HE4 combined detection was 0.911, indicating its clinical diagnostic value was better than that of the two alone. Conclusions In the diagnosis of epithelial ovarian cancer, the specificity of serum HE4 is higher than that of CA125, but the sensitivity is lower than that of CA125. The combined detection of HE4 and CA125 is more conducive to improve the diagnostic accuracy of ovarian epithelial cancer.
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Epithelial ovarian cancer (EOC) has the highest mortality rate among the gynecological malignancies. Because of its early symptoms are not obvious and lack of effective screening methods, more than 70％ patients have been diagnosed at late stage (stage Ⅲ-Ⅳ). Although the 5-year progression-free survival (PFS) of EOC was prolonged with the improvement of treatment methods, its prognosis was still poor, and 50％-95％ patients relapsed within 2 years after treatment. Human epididymis protein 4 (HE4) and carbohydrate antigen 125 (CA125) are widely used in the evaluation of diagnosis, treatment efficacy and prognosis of EOC, but the evaluation effect of both HE4 and CA125 is rarely reported. Therefore, it is necessary to summarize the research progress of HE4 and CA125 in evaluating the diagnosis, treatment efficacy and prognosis of EOC.
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Objective To investigate the clinical efficacy of acupuncture at gastrointestinal Back-Shu and Front-Mu points for post-stroke functional constipation. Method Sixty-six patients with post-stroke functional constipation were randomized to treatment and control groups, 33 cases each. The treatment group received acupuncture at gastrointestinal Back-Shu and Front-Mu points: Weishu (BL21), Dachangshu (BL25), Xiaochangshu (BL27), Zhongwan (CV12), Tianshu (ST25) and Guanyuan (CV4). The control group took Chinese herbal medicine Liumo Decoction prescribed originally. The clinical therapeutic effects were compared between the two groups. The CCS score was recorded and fecal characteristics were scored in the patients at baseline, during treatment and at follow-up. Result The total efficacy rate was 90.9％ in the treatment group and 42.4％ in the control group. The CCS score and the fecal characteristic score were lower in the two groups after treatment compared with before (P<0.05) and the therapeutic effect was more marked in the treatment group (P<0.05). The CCS score and the fecal characteristic score increased in the two groups at follow-up, but the increments were significantly smaller in the treatment group than in the control group (P<0.05). Conclusion Shu-Mu point combination acupuncture can significantly relieve the symptoms of post-stroke functional constipation and change fecal characteristics. It is superior to Chinese herbal treatment alone.
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This paper was aimed to study the effects of icariin (ICA) on the proliferation of vascular smooth muscle cell (VSMC) induced by oxidized low density lipoprotein (ox-LDL), and the molecular mechanism of the expression of proliferating cell nuclear antigen (PCNA) and MAPK signaling pathway. In this study, VSMC was induced by ox-LDL (50 mg•L⁻¹),the effect of ICA on the proliferation of VSMC was detected by MTT assay, Western blot and Real-time PCR. The results showed that after stimulation of ox-LDL, the proliferation activity of VSMC was increased, S phase, G₂/M phase cells were increased, G₀/G₁ phase cells were decreased, PCNA protein expression was enhanced; ICA (40, 20, 10 μmol•L⁻¹) could effectively inhibit ox-LDL-induced VSMC proliferation, S phase and G₂/M phase cells were decreased, the percentage of cells in G₀/G₁ phase were increased, PCNA expression was decreased, p38MAPK and ERK1/2 activation were inhibited. These results indicate that ICA can inhibit the proliferation of VSMC by reducing the expression of PCNA and blocking the p38MAPK and ERK1/2 signaling pathway.
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To study the therapeutic effect and possible mechanism of icariin on myocardial ischemia-reperfusion injury ( MIRI) model in diabetes rats. The model of diabetic rats were induced by Streptozotocin (STZ), then the model of MIRI was established by ligating the reversible left anterior descending coronary artery for 30 min, and then reperfusing for 120 min. totally 40 male SD were randomly divided into five groups: the control group (NS), the ischemia reperfusion group (NIR), the diabetes control group (MS), the diabetic ischemia reperfusion group (MIR) and the diabetic ischemia reperfusion with icariin group (MIRI). The changes in blood glucose, body weight and living status were observed; the enzyme activity of serum CK-MB, LDH, GSH-Px and myocardium SOD and the content MDA and NO in myocardium were detected; the myocardial pathological changes were observed by HE staining; the myocardial Caspase-3, the Bcl-2, Bax protein expressions were detected by Western blot. The result showed that the diabetes model was successfully replicated; myocardial ischemia-reperfusion injury was more serious in diabetes rats; icariin can increase NO, SOD, GSH-Px, Bcl-2 protein expression, decrease MDA formation, CK-MB and LDH activities and Caspase-3 and Bcl-2 protein expressions and myocardial damage. The result suggested that icariin may play a protective role against ischemia reperfusion myocardial injury in diabetes rats by resisting oxidative stress and inhibiting cell apoptosis.
Subject(s)
Animals , Humans , Male , Rats , Apoptosis , Caspase 3 , Metabolism , Creatine Kinase , Metabolism , Diabetes Mellitus, Type 2 , Drugs, Chinese Herbal , Flavonoids , Ischemia , Drug Therapy , Malondialdehyde , Metabolism , Myocardial Reperfusion Injury , Drug Therapy , Metabolism , Myocardium , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Rats, Sprague-Dawley , Superoxide Dismutase , Metabolism , bcl-2-Associated X Protein , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To determine the regulatory role and mechanism of nitric oxide (NO) in the development and hatching of mouse blastocysts.</p><p><b>METHODS</b>The Kunming female mice were superovulated and then mated with mature male mice. On the day 2.5 of their pregnancy, morulae were flushed from their uterine horns with culture media. Morulae were cultured in different concentrations of N-nitro-L arginine methyl ester (L-NAME), sodium nitroprusside (SNP), or the combination of L-NAME and SNP in culture media for 48 hours. The development and hatching of blastocysts were examined on day 4 and day 5 and the total numbers of blastocyst cells and cysteinyl aspartate specific proteinase 3 (caspase 3) were observed under confocal laser scanning microscope.</p><p><b>RESULTS</b>With the increase of the concentration of L-NAME or SNP, the hatching rate of blastocysts and the total number of blastocyst cells were significantly reduced. The addition of 10 nmol/L SNP in culture media with 5 mmol/L L-NAME significantly increased the development of blastocysts and promoted hatching of blastocysts. However, with increase of SNP concentration in culture media with 5 mmol/L L-NAME, the development and hatching rates of blastocysts were significantly decreased. L-NAME had no obvious effect on the expression of active caspase 3 in blastocyst cells. However,when being above 500 nmol/L,SNP significantly increased the expression of caspase 3 in blastocyst cells.</p><p><b>CONCLUSIONS</b>NO plays an important role in development and hatching of mouse blastocysts. Excessively high or low NO can damage the division of blastomeres, resulting in the failure of the blastocyst development and hatching. Also, excessively high NO can lead to the apoptosis of the blastocyst cells.</p>
Subject(s)
Animals , Female , Humans , Male , Mice , Pregnancy , Arginine , Blastocyst , Culture Media , Nitric Oxide , Nitroprusside , UterusABSTRACT
PURPOSE: Near-infrared fluorescence imaging with indocyanine green (ICG) has the potential to improve sentinel lymph node (SLN) mapping in breast cancer. In this clinical trial, we compared the potential value of ICG combined with blue dye with that of blue dye alone for detecting SLNs. METHODS: Patients undergoing SLN biopsy (SLNB) between November 2010 and November 2013 were included. Up to December 2011, SLNs were detected by using patent blue (PB) alone, and since January 2012, by using PB in combination with ICG. The patients were divided into the following two groups: group A (ICG-PB; n=96) and group B (PB; n=73), and SLN detection parameters were compared between the groups. All patients underwent level I and II axillary dissections after SLNB. RESULTS: In group A, the SLN detection rate was 96.9% (93/96), the accuracy of detection was 98.9% (92/93), and the false-negative rate (FNR) was 3.4% (1/29). In group B, the SLN detection rate was 84.9% (62/73), the accuracy of detection was 96.8% (60/62), and the FNR was 11.1% (2/18). The ICG-PB group showed significantly superior results compared to the PB group for SLN detection (p=0.005) and a greatly improved FNR. CONCLUSION: The combined fluorescence and blue dye-based tracer technique was superior to the use of blue dye alone for identifying SLNs, and for predicting axillary lymph node status in patients with breast cancer; in addition, the combined technique had reduced false-negative results.
Subject(s)
Humans , Biopsy , Breast Neoplasms , Fluorescence , Indocyanine Green , Lymph Nodes , Optical Imaging , Sentinel Lymph Node BiopsyABSTRACT
<p><b>OBJECTIVE</b>The aim of this study was to investigate the mRNA expression levels of human leukocyte antigen Class I at different progressive stages of human oral squamous cell carcinomas.</p><p><b>METHODS</b>The expression of mRNA of human leukocyte antigen--B/C was detected in 23 primary tumors, 10 metastatic focuses and 11 histological normal oral epithelia using in situ hybridization method with a digoxigenin--labeled DNA probe. The probe was human leukocyte antigen--B/C locus specific.</p><p><b>RESULTS</b>The hybridization signals were present in the cytoplasm of either normal epithelia or tumor cells. The integrated optical density values of the hybridization signals were detected with the aid of an image analysis system. The results showed that the average integrated optical density values of the primary tumors were statistically lower than the normal oral epithelia (P < 0.05), but there was no significant difference between metastatic tumors and the primary tumors or the normal epithelia. The integrated optical density values measured in the metastatic tumors also did not show statistically differences compared with the primary tumors of the same patients.</p><p><b>CONCLUSION</b>Impaired regulation of human leukocyte antigen--B/C transcription could occur but might not be directly associated with metastasis of oral squamous cell carcinomas.</p>
Subject(s)
Humans , Carcinoma, Squamous Cell , Genetics , Allergy and Immunology , HLA-B Antigens , Genetics , HLA-C Antigens , Genetics , Histocompatibility Antigens Class I , Genetics , In Situ Hybridization , Mouth Neoplasms , Genetics , Allergy and Immunology , RNA, Messenger , Genetics , Transcription, GeneticABSTRACT
<p><b>OBJECTIVE</b>The purpose of this study was to investigate the transcription and expression levels of human leukocyte antigen-DR at different stages of oral squamous cell carcinomas.</p><p><b>METHODS</b>The specific monoclonal antibody and beta-locus specific oligonucleotide probes of human leukocyte antigen-DR were employed in this study. A total of 32 primary oral squamous cell carcinomas, 15 metastatic focuses and 26 histologically normal oral epithelia, were detected for the presence of the human leukocyte antigen-DR molecule by using immunohistochemistry and in situ hybridization.</p><p><b>RESULTS</b>The human leukocyte antigen-DR expression of the primary focuses was significantly higher than that of the normal epithelia (P < 0.05), but their expression did not show statistically difference between the metastatic focuses and the normal epithelia. The immunohistochemistric results were identical with those of in situ hybridization.</p><p><b>CONCLUSION</b>The abnormal higher expression of the HLA-DR is a common character of primary oral squamous cell carcinomas, but it may be not relevant to the metastasis of tumors.</p>
Subject(s)
Humans , Carcinoma, Squamous Cell , Genetics , Allergy and Immunology , HLA-DR Antigens , Genetics , Immunohistochemistry , In Situ Hybridization , Mouth Mucosa , Allergy and Immunology , Mouth Neoplasms , Genetics , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>The purpose of this study was to evaluate the advantages and disadvantages of vascularized free fibula flap as a new method for mandibular reconstruction.</p><p><b>METHODS</b>25 cases (17 male to 8 female) who have received mandibular reconstruction with free vascularized fibular flaps in our hospital were studied retrospectively. The average length of the fibula grafts is 10.0 cm (range from 5.5 to 16 cm). 3 cases received primary insertion of osteointegrated dental implants into the free fibula flap, and all these 5 implants survived.</p><p><b>RESULTS</b>All flaps except 1 were viable. 62% of the cases took normal diet postoperatively, and the remainder took soft diet as well. All patients spoke clearly. No ankle unstability was reported. And the aesthetic assessments in all patients were good or fair.</p><p><b>CONCLUSION</b>Vascularized free fibular flap takes its distinct advantages to other autogeneous free bone flaps and is confirmed to be one of the optimal methods for mandible reconstruction by our study.</p>