[Blaps rynchopetera combined with cyclophosphamide affects proliferation and apoptosis of lung cancer cells via Wnt/ß-catenin signaling pathway].

This study aims to investigate the effects of Blaps rynchopetera Fairmaire and/or cyclophosphamide on the proliferation and apoptosis of lung cancer cells and decipher the underlying mechanism. B. rynchopetera and cyclophosphamide-containing serum and blank serum were prepared from SD rats. Cell counting kit-8(CCK-8) assay was employed to examine the proliferation of lung cancer cell lines A549 and Lewis treated with corresponding agents. The Jin's formula method was used to evaluate the combined effect of the two drugs. According to the evaluation results, appropriate drug concentrations and lung cancer cell line were selected for subsequent experiments, which included control, B. rynchopetera, cyclophosphamide, B. rynchopetera + cyclophosphamide, and B. rynchopetera + Wnt/ß-catenin pathway agonist lithium chloride(LiCl) groups. Immunocytochemistry was employed to measure the expression of proliferation-related proteins in Lewis cells after drug interventions. Flow cytometry was employed to determine the cell cycle and apoptosis. The expression levels of proliferating cell nuclear antigen(PCNA), cyclinD1, B-cell lymphoma 2(Bcl-2), Bcl-2-assiocated X protein(Bax), Wnt1, and ß-catenin were determined by Western blot. The results showed that B. rynchopetera and/or cyclophosphamide significantly inhibited the proliferation of A549 and Lewis cells. Compared with B. rynchopetera alone, the combination increased the inhibition rate on cell proliferation. The combination of B. rynchopetera and cyclophosphamide demonstrated a synergistic effect according to Jin's formula-based evaluation. Compared with the control group, the B. rynchopetera, cyclophosphamide, and B. rynchopetera + cyclophosphamide groups showed increased proportion of Lewis cells in G_0/G_1 phase, increased apoptosis rate, up-regulated expression of Bax, and down-regulated expression of PCNA, cyclinD1, Bcl-2, Wnt1, and ß-catenin. Compared with the cyclophosphamide group, the combination group showed increased proportion of cells in G_0/G_1 phase, increased apoptosis rate, up-regulated expression of Bax, and down-regulated expression of PCNA, cyclinD1, Bcl-2, Wnt1, and ß-catenin. Compared with the B. rynchopetera group, the B. rynchopetera + LiCl group had deceased proportion of cells in G_0/G_1 phase, decreased apoptosis rate, down-regulated expression of Bax, and up-regulated expression of PCNA, cyclinD1, Bcl-2, Wnt1, and ß-catenin. The results indicated that B. rynchopetera could inhibit the proliferation, arrest the cell cycle, and induce the apoptosis of lung cancer cells by inhibiting the Wnt/ß-catenin signaling pathway. Moreover, B. rynchopetera had a synergistic effect with cyclophosphamide.

[Blaps rynchopetera affects proliferation, migration, and invasion of non-small cell lung cancer: a study based on network pharmacology and in vivo and in vitro experiments].

Network pharmacology, molecular docking, and in vivo and in vitro experiments were employed to study the molecular mechanism of Blaps rynchopetera Fairmaire in the treatment of non-small cell lung cancer(NSCLC). The components of B. rynchopetera were collected by literature review, and the active components were screened out through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). PharmMapper was used to obtain the targets of the active components. The targets of NSCLC were obtained from DrugBank, GeneCards, OMIM, TTD, and PharmGKB. The Venn diagram was drawn to identify the common targets shared by the active components of B. rynchopetera and NSCLC. The "drug component-target" network and protein-protein interaction(PPI) network were constructed by Cytoscape, and the key targets were screened by Centiscape. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment of the above key targets were performed by DAVID. AutoDock and PyMOL were used for the molecular docking between the key targets and corresponding active components. A total of 31 active components, 72 potential targets, and 11 key targets of B. rynchopetera against NSCLC were obtained. The active components of B. rynchopetera had good binding activity with key targets. Further, the serum containing B. rynchopetera was prepared and used to culture human lung adenocarcinoma A549 cells. The CCK-8 assay was employed to determine the inhibition rates on the growth of A549 cells in blank control group and those exposed to different concentrations of B. rynchopetera-containing serum, cisplatin, and drug combination(B. rynchopetera-containing serum+cisplatin) for different time periods. The cell migration and invasion of A549 cells were detected by cell scratch assay and Transwell assay, respectively. Western blot was employed to determine the expression levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax), caspase-3, cell division cycle 42(CDC42), proto-oncogene tyrosine-protein kinase SRC, and vascular endothelial growth factor(VEGF) in A549 cells. C57BL/6 mice were inoculated with Lewis cells and randomly assigned into a model control group, a B. rynchopetera group, a cisplatin group, and a drug combination(B. rynchopetera+cisplatin) group, with 12 mice per group. The body weight and the long diameter(a) and short diameter(b) of the tumor were monitored every other day during treatment, and the tumor volume(mm~3) was calculated as 0.52ab~2. After 14 days of continuous medication, the mice were sacrificed for the collection of tumor, spleen, and thymus, and the tumor inhibition rate and immune organ indexes were calculated. The tissue morphology of tumors was observed by hematoxylin-eosin(HE) staining, and the positive expression of Bax, Bcl-2, caspase-3, CDC42, SRC, and VEGF in the tumor tissue was detected by immunohistochemistry. The results indicated that B. rynchopetera and the drug combination regulated the expression levels of Bax, Bcl-2, caspase-3, CDC42, SRC, and VEGF to inhibit the proliferation, migration, and invasion of A549 cells and Lewis cells, thus playing a role in the treatment of NSCLC via multiple ways.

[Effect of acupuncture for dysphagia after stroke based on fiberoptic endoscopic swallowing function evaluation].

OBJECTIVE: To observe the effect of acupuncture combined with regular treatment and swallowing function training on pharyngeal motor, sensory function and penetration-aspiration function in patients with dysphagia after stroke. METHODS: A total of 60 patients with dysphagia after stroke were randomly divided into a control group and an observation group, 30 patients in each group. Both groups were treated with conventional treatment and swallowing function training; in addition, the observation group was treated with acupuncture at Lianquan (CV 23), Fengfu (GV 16), Yifeng (TE 17). All the treatments were given once a day, 5 days a week, for totally 4 weeks. In the two groups, the pharyngeal motor and sensory function, penetration-aspiration scores were evaluated by fiberoptic endoscopic evaluation of swallowing (FEES), and the Kubota water swallowing test scores were assessed before and after treatment, and the clinical effects were compared. RESULTS: After treatment, the pharyngeal motor and sensory function in the two groups were all higher than those before treatment (P<0.05), and those in the observation group were better than the control group (P<0.05). After treatment, the penetration-aspiration scores and Kubota water swallowing test scores in the two groups were all lower than those before treatment (P<0.05), and those in the observation group were lower than the control group (P<0.05). The total effective rate was 93.3% (28/30) in the observation group, which was better than 73.3% (22/30) in the control group (P<0.05). CONCLUSION: Acupuncture combined with regular treatment and swallowing training could improve the pharyngeal motor and sensory function, and penetration-aspiration scores in patients with dysphagia after stroke.

[Non-real-time endobronchial bronchoscopy ultrasound assisted transbronchial lung biopsy in diagnosing peripheral pulmonary lesions].

OBJECTIVE: To evaluate the role of non-real-time endobronchial bronchoscopy ultrasound(EBUS) assisted transbronchial lung biopsy (TBLB) in diagnosing peripheral pulmonary lesions (PPL). METHODS: One hundred and five patients [68 males and 37 females, mean age (59 ± 12) years, ranged from 39 - 81 years] with PPL confirmed by computered tomography (CT) were recruited in this study between June 1st 2011 and March 1st 2012. All cases received bronchoscopy examinations and presented with roughly normal results. Fifty-four cases received EBUS examinations. For peripheral lesions with accessible EBUS images, blind biopsy was performed with biopsy forceps through pathways of the ultrasonic probe after the retreat of the probe. In those cases without accessible EBUS images, blind biopsy was performed based on the localization by image data. The other 51 cases without EBUS testing underwent blind biopsy on the localization by image data. Positive rates of pathological diagnosis of the 2 groups were compared. Analysis was by χ(2)-test. RESULTS: In 54 patients who received EBUS examinations, 76% (41/54) of PPLs were detected performed by EBUS. The positive rate of the EBUS assisted TBLB group was 67% (36/54), compared with 45% (23/51) in the general TBLB group. There was a better diagnostic rate (P < 0.05) in the EBUS assisted TBLB group than the general TBLB group. Thirteen patients without accessible EBUS images obtained negative pathological results. The diagnosis rate of EBUS assisted TBLB on lesions with ≤ 30 mm minimum diameter was 44% (8/18), lower than 78% (28/36) on lesions with > 30 mm minimum diameter (P < 0.05). In terms of diagnosis rate on lesions with ≤ 30 mm minimum diameter, EBUS assisted TBLB was 44% (8/18), higher than 12% (2/17) of TBLB alone (P < 0.05). As for lesions with > 30 mm minimum diameter, diagnosis rate of EBUS assisted TBLB was 52% (28/54) and TBLB alone was 41% (21/51), representing insignificant difference (P > 0.05). In the EBUS assisted TBLB group, we performed 269 blind biopsies, with an average of 4.8 times per case, whereas the general TBLB group required 398 times, with an average of 7.8 times per case. EBUS assisted TBLB decreased the operation times of blind biopsy (P < 0.05) to acquire adequate and appropriate specimen. Complications of biopsy occurred in this study included slight haemoptysis (61/105, 58.1%), chest pain (25/105, 23.8%) and pneumothorax (2/105, 1.9%). Patients with these complications recovered spontaneously without special managements. CONCLUSIONS: Non-real-time EBUS assisted TBLB could improve diagnostic positive rate without increasing operational risk. In most cases, the blind biopsy did not succeed if EBUS failed to detect the lesions. The success rate of non-real-time EBUS assisted TBLB was related to the minimum diameter of PPL. In terms of diagnosis rate on lesions with ≤ 30 mm minimum diameter, EBUS assisted TBLB was higher than TBLB alone. As for lesions with >30mm minimum diameter, there was no significant difference in the diagnosis rate between these 2 groups. EBUS assisted TBLB decreased the times of blind biopsy process (P < 0.05) to obtain adequate and appropriate specimen.

[Quantitative analysis of the ultrasonogram and histological findings in normal airway wall].

OBJECTIVE: To find out the correlation between endobronchial ultrasonography (EBUS) images and histologic findings in normal bronchial wall via quantitative analysis of the airway wall thickness and the layer thickness. METHODS: From July 1st to December 31th in 2010, patients underwent lobectomy performed endobronchial ultrasonography (EBUS) before surgery and frost pathological examination after surgery. The layer thickness of EBUS and pathological images were measured. Bland-Altman plots were used to analyze the agreement between EBUS measurements and pathological measurements. RESULTS: Twenty-one patients were enrolled in the study. Five layers of the wall were distinguished at the ultrasonogram. Starting on the luminal side, the first, third and fifth layer (L1, L3, L5) were hyperechoic while the second, fourth layer (L2, L4) were hypoechoic. The wall thickness with good agreement was almost equal between the 2 kinds of images (1.877:1.745). L1 thickness was lager than the mucosa thickness (0.275:0.164). L2 thickness was smaller than the submucosa thickness (0.100:0.202). L1 + L2 thickness was almost equal to the thickness of mucosa and submucosa layer (0.375:0.366). The Bland-Altman plots showed poor agreement between the L1, L2 thickness and the mucosa thickness, the submucosa thickness while good agreement between the L1 + L2 thickness and the thickness of mucosa and submucosa layer. L3 thickness was lager than the inner perichondrium thickness (0.241:0.075), and L4 thickness was smaller than the cartilage layer thickness (0.655:0.811). L3 + L4 thickness was almost equal to thickness of the inner perichondrium and the cartilage layer (0.895:0.887). L5 thickness was almost equal to thickness of the outer perichondrium and the connective tissue outside the cartilage layer (0.533:0.491). The Bland-Altman plots showed poor agreement between the L3, L4 thickness and the inner perichondrium thickness, cartilage layer thickness, while good agreement between L5, L3 + L4, L3 + L4 + L5 thickness and the corresponding indexes. CONCLUSIONS: There is a five-layer structure on the bronchial EBUS image including the first layer at the luminal side corresponding to the mucosa and inner part of the submucosa; the second layer corresponding to the outer part of submucosal tissue; the third layer corresponding to the inner perichondrium and the inner part of the cartilage; the fourth layer corresponding to the outer part of cartilage; the fifth layer corresponding to the outer perichondrium and the connective tissue outside the cartilage layer.

[Transcriptional differences between a heterokaryon and its segregants of Fusarium oxysporum f. sp. vasinfectum].

In order to elucidate the mechanism of fungal heterokayosis, a wild type strain of Fusarium oxysporum f. sp. vasinfectum was isolated from the cotton field in Anyang, Henan Province. Through single hyphal-tip isolation, a heterokaryon, Ag149, was obtained, and its two different phenotypic segregants, Ag149-I and Ag149-III, were separated from the mutated sectors on colony of the heterokaryon. They have remarkable differences on color of colony, morphology of hypha and pathogenicity. After analyzing by RAPD on their nuclear DNA with 100 random primers, no polymorphic difference was found among them. On going to find the different expressed gene, the mRNA differential display method was performed. Two kinds of reverse transcriptase AMV and MMLV, and a kit which consists of three kinds of 3' terminal anchor primers and eight kinds of 5' terminal arbitrary primers were used in differential display PCR (DD-PCR). Total RNA as template was reverse transcribed into corresponding cDNA by 3' terminal anchor primers, and the cDNA were amplified by polymerase chain reaction with a set of one same 3' anchor primer and one 5' arbitrary primer. The PCR products were then resolved on denaturing polyacylamimide gel, and the cDNA bands were visualized by silver staining. Among the 144 PCR products, 19 differentially expressed cDNA fragments ranged from 300 bp to 700 bp were purified. All of them were ligated to pGEM-T vector respectively for sequencing and Rev-Northern blotting. Two cDNA fragments (G5 and C6) were observed to be positive after Rev-Northern blotting. The C6 was highly expressed in the heterokaryon Ag149 and its segregant Ag149-I. It is 564 bp in length and can be predicted 77 amino acids from the beginning of the 3rd to 233th base, and then searched from GenBank. The amino acid sequence of C6 shared homologies with the 6th subunit of NADH dehydrogenase found in some bacteria, plants and animals at 30% - 70% level. While the G5 was highly expressed in Ag149 and its segregant Ag149-III. It is 432 bp in length and can be predicted 101 amino acids from the beginning of the 2nd to 304th base, and the amino acid sequence is 35% homologies comparing with the tetracycline efflux protein (OtrB) of Streptomyces rimosus. We also checked these two kinds of the pGEM-T vector harboring cDNA fragments (C6 and G5) by Southern blotting with their nuclear DNA and mitochondrial DNA (mtDNA) separately. The positive identification signals only appeared from nuclear DNA,and it addressed that C6 and G5 locate on their nuclear genome. The result indicated that the difference between the heterokaryon and its segregants is distinct on gene transcriptional level. Thus a molecular evidence for the formation of heterokaryon in filamentous fungi was provided.