OBJECTIVE: To explore the role of the natural compound hesperidin in glycolysis, the key ratelimiting enzyme, in colorectal cancer (CRC) cell lines. METHODS: In vitro, HCT116 and SW620 were treated with different doses of hesperidin (0-500 µmol/L), cell counting kit-8 and colone formation assays were utilized to detected inhibition effect of hesperidin on CRC cell lines. Transwell and wound healing assays were performed to detect the ability of hesperidin (0, 25, 50 and 75 µmol/L) to migrate CRC cells. To confirm the apoptotic-inducing effect of hesperidin, apoptosis and cycle assays were employed. Western blot, glucose uptake, and lactate production determination measurements were applied to determine inhibitory effects of hesperidin (0, 25 and 50 µmol/L) on glycolysis. In vivo, according to the random number table method, nude mice with successful tumor loading were randomly divided into vehicle, low-dose hesperidin (20 mg/kg) and high-dose hesperidin (60 mg/kg) groups, with 6 mice in each group. The body weights and tumor volumes of mice were recorded during 4-week treatment. The expression of key glycolysis rate-limiting enzymes was determined using Western blot, and glucose uptake and lactate production were assessed. Finally, protein interactions were probed with DirectDIA Quantitative Proteomics, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. RESULTS: Hesperidin could inhibit CRC cell line growth (P<0.05 or P<0.01). Moreover, hesperidin presented an inhibitory effect on the migrating abilities of CRC cells. Hesperidin also promoted apoptosis and cell cycle alterations (P<0.05). The immunoblotting results manifested that hesperidin decreased the levels of hexokinase 2, glucose transporter protein 1 (GLUT1), GLUT3, L-lactate dehydrogenase A, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), PFKFB3, and pyruvate kinase isozymes M2 (P<0.01). It remarkably suppressed tumor xenograft growth in nude mice. GO and KEGG analyses showed that hesperidin treatment altered metabolic function. CONCLUSION Hesperidin inhibits glycolysis and is a potential therapeutic choice for CRC treatment.
Hesperidin/therapeutic use* ; Colorectal Neoplasms/metabolism* ; Glycolysis/drug effects* ; Animals ; Humans ; Apoptosis/drug effects* ; Mice, Nude ; Cell Movement/drug effects* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Glucose/metabolism* ; Cell Cycle/drug effects* ; Mice, Inbred BALB C ; Mice ; HCT116 Cells ; Lactic Acid

Objective To observe the clinical efficacy of successive trigger needling combined with fire needling in the treatment of rheumatoid arthritis of cold-damp obstruction type.Methods A total of 72 patients with rheumatoid arthritis of cold-damp obstruction type were randomly divided into observation group and control group,36 cases in each group.The control group was treated with fire needling combined with ordinary acupuncture,and the observation group was treated with fire needling combined with filiform needle.The treatments were performed once every other day,3 times a week,2 weeks as a course of treatment,continuous treatment for 2 courses.After 4 weeks of treatment,the clinical efficacy of the two groups was evaluated.The changes of pain Visual Analogue Scale(VAS)score and Self-Rating Anxiety Scale(SAS)score before and after treatment,and the time of morning stiffness of the joints were observed in the two groups.The changes of the number of joint swelling and the number of joint tenderness were compared before and after treatment between the two groups.Results(1)After treatment,the time of morning stiffness of the joints of the two groups of patients was significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving morning stiffness of the joints,and the difference was statistically significant(P＜0.05).(2)After treatment,the number of swollen joints and the number of joint pressure and pain in the two groups were significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving swollen joints and joint pressure and pain,and the difference was statistically significant(P＜0.05).(3)After treatment,the VAS and SAS scores of patients in the two groups were significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving the VAS and SAS scores,and the difference was statistically significant(P＜0.05).(4)The total effective rate of the observation group was 91.66%(33/36),while that of the control group was 77.78%(28/36).The efficacy of the observation group was superior to that of the control group,and the difference was statistically significant(P＜0.05).Conclusion Successive trigger needling combined with fire needling treating rheumatoid arthritis of cold-damp obstruction type can significantly improve the clinical symptoms of patients,reduce their anxiety,and thus improve the quality of life of patients,with remarkable efficacy.

Objective: To analyze the phenotype and genotype of two pedigrees with inherited fibrinogen (Fg) deficiency caused by two heterozygous mutations. We also preliminarily probed the molecular pathogenesis. Methods: The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and plasma fibrinogen activity (Fg∶C) of all family members (nine people across three generations and three people across two generations) were measured by the clotting method. Fibrinogen antigen (Fg:Ag) was measured by immunoturbidimetry. Direct DNA sequencing was performed to analyze all exons, flanking sequences, and mutated sites of FGA, FGB, and FGG for all members. Thrombin-catalyzed fibrinogen polymerization was performed. ClustalX 2.1 software was used to analyze the conservatism of the mutated sites. MutationTaster, PolyPhen-2, PROVEAN, SIFT, and LRT online bioinformatics software were applied to predict pathogenicity. Swiss PDB Viewer 4.0.1 was used to analyze the changes in protein spatial structure and molecular forces before and after mutation. Results: The Fg∶C of two probands decreased (1.28 g/L and 0.98 g/L, respectively). The Fg∶Ag of proband 1 was in the normal range of 2.20 g/L, while it was decreased to 1.01 g/L in proband 2. Through genetic analysis, we identified a heterozygous missense mutation (c.293C>A; p.BβAla98Asp) in exon 2 of proband 1 and a heterozygous nonsense mutation (c.1418C>G; p.BβSer473*) in exon 8 of proband 2. The conservatism analysis revealed that Ala98 and Ser473 presented different conservative states among homologous species. Online bioinformatics software predicted that p.BβAla98Asp and p.BβSer473* were pathogenic. Protein models demonstrated that the p.BβAla98Asp mutation influenced hydrogen bonds between amino acids, and the p.BβSer473* mutation resulted in protein truncation. Conclusion: The dysfibrinogenemia of proband 1 and the hypofibrinogenemia of proband 2 appeared to be related to the p.BβAla98Asp heterozygous missense mutation and the p.BβSer473* heterozygous nonsense mutation, respectively. This is the first ever report of these mutations.
Humans ; Afibrinogenemia/genetics* ; Codon, Nonsense ; Pedigree ; Phenotype ; Fibrinogen/genetics* ; Genotype

The aim of this paper was to investigate the preventive effects of Keluoxin Capsules(KLX) on diabetic retinopathy in db/db mice. One hundred male db/db diabetic mice(45-55 g, 8 weeks) were randomly divided into 5 groups(model, KLX low dose, KLX middle dose, KLX high dose, Dobesilate) and 20 male C57 BL/KsJdb~(+/+) were taken as control group. Body weight and fasting blood-glucose were detected every week. Mice were administrated with saline(control and model group), KLX(780, 1 560, 3 120 mg·kg~(-1)·d~(-1), ig), Dobesilate(195 mg·kg~(-1)·d~(-1), ig) for 20 weeks, respectively. At the end of the administration, optical coherence tomography, fundus fluorescein angiography and electroretinogram of the retina were measured. The eyeball was extirpated and retina was isolated to make paraffin section, followed by HE staining and glial fibrillary acidic protein(GFAP) immunohistochemistry. The results indicated that KLX has no obvious effect on body weight and fasting blood level in db/db mice. However, KLX could significantly regulate the thickness of retinal ganglion layer and inner plexiform layer. KLX was able to remarkably reduce the quantity of diabetic microvessel. Meanwhile, KLX could notably improve retinal function. Moreover, KLX could observably modulate the cell arrangement and edema in each layer. There was no markable difference in retina according to the immunochemistry assay. In the present study, KLX exert marked preventive effects on diabetic retinopathy in db/db mice, which provided an experimental evidence for clinical use.
Animals ; Capsules ; Diabetes Mellitus, Experimental ; Diabetic Retinopathy ; drug therapy ; Fluorescein Angiography ; Hypoglycemic Agents ; pharmacology ; Male ; Mice ; Random Allocation ; Retina ; drug effects