OBJECTIVETo establish and evaluate a BA-ELISA method for the quantitative detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein.

METHODSWe deliberately selected three tables of CFTR and made the synthetic peptide be expressed in E. coli, then used the antigen to immunize rabbits to obtain the anti-CFTR polyclonal serum. After that, 96 well plates were coated with the purified antibody against CFTR. The antigen CFTR which was extracted from human sperm was detected by anti-CFTR antibody labeled with biotin, horseradish peroxidase conjugated avidin, and the substrate. The concentrations of two kinds of antibodies and the experiment parameters were optimized. Thereby, the double antibody sandwich BA-ELISA method for the quantitative detection of CFTR protein was established. Furthermore, the reproducibility, specificity and so on were evaluated by clinical specimens of sperm.

RESULTSThe optimal concentration of coated anti-CFTR IgG was 4 µg/ml, while the biotin labeled anti-CFTR IgG was 10 µg/ml; the optimal blocking buffer was 1% BSA-PBST, the optimal time of the reaction between antigen and antibody was 60 min, the optimal chromogenic time was 15 min, the intra-assay and inter-assay coefficient were 2.16%-9.23% and 2.29%-11.71% respectively; The lowest detectable limit was 0.15 ng/ml; the standard curve had a good linear correlation of R2 = 0.962.

CONCLUSIONThe BA-ELISA method for the quantitative detection of CTFR protein is successfully established, and it is demonstrated that the method has strong specificity, high sensitivity and good reproducibility. It provides the basis and evidence of the further application of the method.

Animals ; Antibodies ; Cystic Fibrosis Transmembrane Conductance Regulator ; analysis ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; Humans ; Peptides ; Rabbits ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVETo investigate the role of Xuebijing injection(XBJI, traditional Chinese medicine), in inhibiting TLR4--NF-kappaB--IL-1beta pathway of myocardial hypoxia/reoxygenation in rats.

METHODSThirty six male SD rats (280 +/- 30) g were randomly divided into six groups (n = 6): normal group (N group), balanced perfusion group (BP group), model group (M group), low dose XBJI group (XBJI(L) group), middle dose XBJI group (XBJI(M) group), high dose XBJI group (XBJI(H) group). By Langendorff isolated heart perfusion device to establish the model of myocardial hypoxia/reoxygenation in rats. ELISA was used to detect the concentration of interleukin-1beta (IL-1beta); Western blot was used to detect the expression of nuclear factor kappa B p65 (NF-kappaB p65) protein and toll like receptor 4 (TLR4) protein; and RT-PCR to determine the expression of NF-kappaB p65 mRNA and TLR4 mRNA;To observe microstructure changes of hypoxia/reoxygenation myocardial by light microscopy.

RESULTSCompared with M group, the IL-1beta concentration, NF-kappaB p65 and TLR4 protein,NF-kappaB p65 and TLR4 mRNA of XBJIL group, XBJI(M) group, XBJI(H) group expression decreased in varying degrees,and decreased most obviously all in XBJI(M) group (P < 0.05, P < 0.01); Myocardical structural damage was serious in M group, and improved after treatment XBJI, the most obvious was the XBJI(M).

CONCLUSIONDifferent dose of XBJI can inhibit TLR4--NF-kappaB--IL-1beta signal transduction pathway and reduce several inflammatory reaction after myocardial hypoxia/reoxygenation injury, the 4 ml/100 ml of XBJI is the best.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Heart ; drug effects ; Inflammation ; Interleukin-1beta ; metabolism ; Male ; Myocardium ; pathology ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; drug therapy ; Signal Transduction ; Toll-Like Receptor 4 ; metabolism ; Transcription Factor RelA ; metabolism

OBJECTIVETo explore the role of Xuebijing Injection (XBJI) in inhibiting inflammatory factors associated with anoxia/reoxygenation myocardial inflammatory response of rats.

METHODSTotally 36 healthy male Sprague-Dawley rats, 280 ± 30 g were randomly divided into six groups, i.e., the normal control group (N group), the balanced perfusion group (BP group),the model group (M group),the low dose XBJI group (XBJI(L) group), the middle dose XBJI group (XBJI(M) group),and the high dose XBJI group (XBJI(H) group), 6 in each group. The myocardial anoxia/reoxygenation rat model was established by Langendorff isolated heart perfusion. The concentration of TNF-α in the myocardial tissue was detected by ELISA. The expression of nuclear factor kappa B p65 (NF-κB p65) protein and Toll like receptor 4 (TLR4) protein were detected using Western blot. The expression of NF-κB p65 mRNA and TLR4 mRNA was detected by RT-PCR. Ultrastructural changes of anoxia-reoxygenation rats' heart muscle were observed under transmission electron microscope.

RESULTSCompared with the M group,the TNF-α concentration, expression levels of NF-κB p65 protein and mRNA, TLR4 protein and mRNA decreased to various degrees in the XBJI(L) group, the XBJI(M) group, and the XBJI(H) group. The TNF-α expression level decreased most significantly in the XBJI(L), group (P < 0.01), while other indices decreased most obviously in the XBJI(M) group (P < 0.01, P < 0.05). Expression levels of NF-κB p65 and TLR4 protein were obviously lower in the XBJI(M) group than in the XBJI(L) group (P < 0.05). There was no statistical difference in other indices among the three XBJI groups (P > 0.05). Myocardial fibers were loose and broken with disappearance of transverse striation, and mitochondrial cristae was dissolved and severely damaged in the M group. The aforesaid condition was improved after treated by XBJI, with the most obvious effect obtained in the XBJI(M) group.

CONCLUSIONSDifferent doses of XBJI could attenuate inflammatory reactions after myocardial anoxia/reoxygenation rats' heart muscle through inhibiting TLR4-NF-κB-TNF-α signal transduction pathway. The best effect could be obtained by 4 mL/100 mL XBJI.

Animals ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Hypoxia ; Male ; Myocardium ; metabolism ; Myocytes, Cardiac ; NF-kappa B ; metabolism ; Oxygen ; metabolism ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 4 ; metabolism ; Transcription Factor RelA ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the effects of ischemic postconditioning (IPostC) on pneumocyte apoptosis after lung ischemia/reperfusion injury in rats.

METHODSAdult male SD rats were randomly divided into 3 groups based upon the intervention (n = 8): control group (C), lung ischemic reperfusion group (LIR), LIR+ IPostC group (IPostC). At the end of the experiment, blood specimens drawn from the arteria carotis were tested for the content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and myeloperoxidase (MPO); the pneumocyte apoptosis index (AI) was achieved by tennrminal deoxynucleotidyl transferase mediated dUTP nick end abeling (TUNEL); the expression of Bcl-2, Bax protein in lung tissue was accessed by quantitative immunohistochemistry (MHC) and Bcl-2, Bax mRNA by RT-PCR.

RESULTSIPostC could significantly attenuate the MDA level, MPO activity and improve SOD activity in blood serum which was comparable to I/R and significantly reduced the number of TUNEL-positive cells compared with I/R group, expressed as Al (% total nuclei) from (39.0 +/- 3.46) to (8.0 +/- 0.88) (P < 0.01). The protein and mRNA expression of Bcl-2 and Bax showed that IPO significantly attenuated the ischemia/reperfusion-upregulated expression of Bax protein but improved the expression of Bcl-2 that improved the Bcl-2/Bax ratio (P < 0.01) .

CONCLUSIONIPostC may attenuate pneumocyte apoptosis in LIRI by up-regulating expression of Bcl-2/Bax ratio and by inhibiting oxidant generation and neutrophils filtration.

Alveolar Epithelial Cells ; cytology ; Animals ; Apoptosis ; Ischemic Postconditioning ; Lung ; metabolism ; pathology ; Lung Injury ; physiopathology ; Male ; Malondialdehyde ; metabolism ; Peroxidase ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; physiopathology ; Superoxide Dismutase ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo study the effect of Notoginsenoside Rgl on p38 mitogen activated protein kinase (p38MAPK) expression in pulmonary artery smooth muscle cells (PASMCs) cultured in hypoxia hypercapnia.

METHODSSD rat PASMCs was primary cultured, the cells of passage 2- 5 were divided into six groups: normoxic group (N group), hypoxia hypercapnia group (H group), dimethyl sulfoxide (DMSO) control group (HD group), Rg1 treated group (Rg low dose, Rg middle dose and Rg high dose group). Western blot was used to detect the expression of p-p38MAPK protein, and RT-PCR to determine the expression of p38MAPK mRNA.

RESULTSWestern blot and RT-PCR analysis indicated that the expression of p-p38MAPK protein and p-p38MAPK mRNA were significantly higher in HD group than those in N group (P < 0.01). Whereas, in Rg1 treated groups, the level of p-p38MAPK markedly decreased (P < 0.01) in dose-dependent manner.

CONCLUSIONNotoginsenoside Rg1 has protective effects on PASMCs under hypoxia hypercapnia condition, which may be related to inhibiting expression of p38MAPK.

Animals ; Cell Hypoxia ; Cells, Cultured ; Ginsenosides ; pharmacology ; Hypercapnia ; metabolism ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Myocytes, Smooth Muscle ; drug effects ; Pulmonary Artery ; cytology ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases ; metabolism

Animals ; Ginsenosides ; pharmacology ; Hypertension, Pulmonary ; etiology ; metabolism ; Hypoxia ; complications ; metabolism ; Lung ; drug effects ; metabolism ; MAP Kinase Signaling System ; Male ; Panax ; Rats ; Rats, Sprague-Dawley

Immunotherapy is a new strategy for cancer treatment that has the potential to treat all types of cancer. T cell immunoglobulin and mucin-domain-containing molecule-3 (TIM-3) is a key negative regulator of T cell activation. TIM-3 blockage using anti-TIM-3 monoclonal antibody therapy has a great appeal and special advantages. Nanobodies, derived from heavy chain fragment in camelid animals, are now proving clinical values in the development of antibody drugs. In this study, we have immunized camel with TIM-3 antigens and then constructed phage display library. Moreover, 29 nanobodies with different complementarity-determining regions sequences have been screened from the phage display library by phage display technology. In addition, we successfully constructed the cell line stably expressing TIM-3, and screened 10 TIM-3 nanobodies with high specificity and high affinity using flow cytometry. Our study will lay the foundation for the future screening and development of anti-TIM-3 whole humanized functional nanobody.

Epithelial cell adhesion molecule (EpCAM) is a popular target for cancer therapy. In this research, 3 nanobodies with high specificity and endocytosis activity against EpCAM were developed, which provides a basis for the study of immunotoxin based on EpCAM. In our preliminary experiments, we have immunized a camel with EpCAM-Fc antigen and constructed a high-quality phage display library. Seventeen nanobodies with different complementarity determining region (CDR) 3 sequences have been screened after 3 rounds of biopanning by phage display technology. The animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Fudan University School of Pharmacy. After purification, 7 nanobodies showed high cell binding activity by fluorescent activated cell sorting (FACS) identification. Furthermore, 3 nanobodies presented high endocytosis activity based on FACS and laser confocal microscopy, which also showed high affinity to EpCAM measured by ForteBio. According to this study, we aimed to provide a novel alternative approach to the EpCAM-targeted therapy and to provide guidance for the study of nanobody based immunotoxins for other targets.

Objective: To study the effects of dihydromyricetin (DMY) on the proliferation, apoptosis and epithelial mesenchymal transition (EMT) of esophageal squamous cell carcinoma (ESCC) cell KYSE150 and KYSE410. Methods: KYSE150 and KYSE410 cells were treated with different concentrations of DMY (0, 25, 50, 100, 150, 200 μmol/L) for 24 hours. The median inhibition concentration (IC₅₀) values of KYSE150 and KYSE410 were detected by cell counting kit-8 (CCK-8) method. Then 0.5‰ dimethyl sulfoxide (DMSO) was used as control group, dihydromyricetin (DMY), dihydromyricetin and transforming growth factor-β1 (DMY+ TGF-β1), transforming growth factor-β1 (TGF-β1) were used as experimental group. Cell proliferation and apoptosis rates were measured by clonal formation and flow cytometry. Transwell invasion and wound healing assay were used to detect cell invasion and migration. The protein expression levels of Caspase-3, Caspase-9, Bcl-2, Bax, Smad2/3, phosphorylation-Smad2/3 (p-Smad2/3) and Vimentin were detected by western blot. Results: The IC₅₀ values of DMY on KYSE410 and KYSE150 cells were 100.51 and 101.27 μmol/L. The clone formation numbers of KYSE150 and KYSE410 in DMY group [(0.53±0.03) and (0.31±0.03)] were lower than those in DMSO group [(1.00±0.10) and (1.00±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in DMY group [(1.84±0.22)% and (2.80±0.07)%] were higher than those in DMSO group [(1.00±0.18)% and (1.00±0.07)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in DMY group [(0.42±0.03) and (0.29±0.05)] were lower than those in DMSO group [(1.00±0.08) and (1.00±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in DMY group [(0.65±0.14)% and (0.40±0.17)%] were lower than those in DMSO group [(1.00±0.10)% and (1.00±0.08)%, P<0.05]. The clone formation numbers of KYSE150 and KYSE410 in TGF-β1 group [(1.01±0.08) and (0.99±0.25)] were higher than those in DMY+ TGF-β1 group [(0.73±0.10) and (0.58±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in TGF-β1 group [(0.81±0.14)% and (1.18±0.10)%] were lower than those in DMY+ TGF-β1 group [(1.38±0.22)% and (1.85±0.04)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in TGF-β1 group [(1.19±0.11) and (1.39±0.11)] were higher than those in DMY+ TGF-β1 group [(0.93±0.09) and (0.93±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in TGF-β1 group [(1.87±0.19)% and (1.32±0.04)%] were higher than those in DMY+ TGF-β1 group [(0.86±0.16)% and (0.77±0.12)%, P<0.05]. The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY group were higher than those in DMSO group, while the protein expression level of Bcl-2 was lower than that in DMSO group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in DMY group were lower than those in DMSO group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in TGF-β1 group were lower than those in DMY+ TGF-β1 group, and the protein expression level of Bcl-2 was higher than that in DMY+ TGF-β1 group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY+ TGF-β1 group were lower than those in DMY group, and the protein expression level of Bcl-2 was higher than that in DMY group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in TGF-β1 group were higher than those in DMY+ TGF-β1 group (P<0.05). Conclusion: DMY can inhibit the proliferation and EMT of ESCC mediated by TGF-β1 and promote cell apoptosis.
Apoptosis ; Caspase 3/metabolism* ; Caspase 9/metabolism* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Dimethyl Sulfoxide/pharmacology* ; Epithelial-Mesenchymal Transition ; Esophageal Neoplasms/metabolism* ; Esophageal Squamous Cell Carcinoma ; Flavonols ; Humans ; Signal Transduction ; Transforming Growth Factor beta1/pharmacology* ; Vimentin/metabolism* ; bcl-2-Associated X Protein/pharmacology*

Establish a production line with controllable process and high intelligence, contribute to improve the quality and production efficiency of aconite processed by microwave, and promote the transformation and application of aconite processed by microwave. According to the principle of aconite detoxification and the characteristics of industrial microwave equipment, an industrial production line of aconite processed by microwave was established with diester alkaloids and monoester alkaloids as indicators, and pilot production was carried out. At the same time, the content of active constituents and efficacy were compared with that of the main processed products, such as Shengfupian, Baifupian and Heishunpian. The results showed that the industrial production of aconite processed by microwave can be divided into two stages: "Liquid seal to detoxification - drying and puffing". The content of monoester alkaloids in 10 batches of aconite processed by microwave was 0.071%-0.166% and the content of diester alkaloids was 0.004%-0.016%, which met the relevant requirements of the Chinese Pharmacopoeia in 2020. Compared with Heishunpian and Baifupian, the retention rate of the effective components of aconite processed by microwave was higher. Pharmacological experiments showed that aconite processed by microwave not only retained the anti-inflammatory and analgesic activities of Heishunpian and Baifupian, but also significantly increased the levels of leukocytes and lymphocytes in mice with liver cancer chemotherapy, enhanced the CD4/CD8 ratio in spleen cells of mice (P < 0.05), thus regulating the body's immunity. However, this effect of Baifupian was weak, while Heishunpian and Shengfupian had no such effect. Through the above research, this study established microwave processing line with controllable process and high intelligence, as well produced the aconite processed by microwave with low toxicity and stable quality. It laid a foundation for the industrialized continuous production and clinical positioning of aconite by microwave processed, and provided scientific support for the development and application of microwave technology in the field of traditional Chinese medicine. All animal experiments in this study were reviewed and approved by the Experimental Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine before being carried out (Approval No. 2020-28).