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ObjectiveTo explore the mechanism of hypericin against liver cancer using network pharmacology. MethodThe traditional Chinese medicine systems pharmacology database and analysis platform （TCMSP）， Drug Gene Interaction Database （DGIdb）， Comparative Toxicogenomics Database （CTD） and SwissTargetPrediction were used to predict the targets of hypericin. Five databases including GeneCards and Online Mendelian Inheritance in Man （OMIM） were employed to obtain liver cancer-related targets. The intersection was performed to obtain the targets of hypericin against liver cancer. The Database for Annotation， Visualization and Integrated Discovery （DAVID） v2021q4 was used for Gene Ontology （GO） function annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis. The protein-protein interaction （PPI） network of the targets was constructed by Cytoscape 3.6.1 to screen the core targets，and the affinity between hypericin and the core targets was verified by molecular docking. The effects of hypericin on liver cancer and the migration of liver cancer cells were observed by cell viability assay and would healing assay， respectively， and its effects on the mRNA and protein expression of key targets cysteinyl aspartate-specific protease-3（Caspase-3） and mitogen-activated protein kinase 3 （MAPK3） were detected by real-time polymerase chain reaction（Real-time PCR） and Western blot， respectively. ResultA total of 45 genes related to the anti-liver cancer effect of hypericin were obtained， and six core target genes were screened. The signal pathways enriched by KEGG pathway analysis included apoptosis，tumor necrosis factor （TNF） and cancer signal pathways. Molecular docking showed that the core target genes Caspase-3，TNF，estrogen receptor 1 （ESR1），MAPK3，catalase （CAT） and cyclooxygenase 2 （PTGS2） had good affinity with hypericin，especially Caspase-3 and MAPK3. In addition，compared with the conditions in control group， cell experiments demonstrated that hypericin could reduce the viability of liver cancer cells （P<0.05），inhibit their migration，increase the mRNA expression of Caspase-3 （P<0.05） and decrease that of MAPK3 （P<0.05）. ConclusionHypericin exerted the anti-liver cancer effect by affecting the core targets such as Caspase-3，TNF，ESR1，MAPK3，CAT and PTGS2 and jointly interfering with apoptosis，TNF and cancer signal pathways.
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Objective:To explore the changes in somatosensory evoked potential (SEP) in rats with spinal cord injury (SCI) and the effects of relieving spinal cord compression at different times on recovery and the evoked potential.Methods:Seventy Sprague-Dawley rats were randomly divided into a control group of 10 and an experimental group of 60. The experimental group was further divided into a mild injury group of 10, a moderate injury group of 40 and a severe injury group of 10. Spinal cord injuries with different severities were established in the experimental group using a self-made percussion device. The rats′ SEPs were measured before the injury, and 5 minutes, 1 hour, 6 hours, 3 days and 7 days afterward. Some of the rats receiving decompression treatment.Results:The more seriously the spinal cord was injured, the longer was the latency and the smaller was the amplitude. Both differences were statistically significant. Rats with longer compression time had significantly longer incubation periods and greater decreases in the amplitude. After relieving the compression, rats from whom it had been relieved earlier had quicker amplitude recovery. For rats under compression for 30 minutes, their amplitude was the lowest seven days later.Conclusions:For spinal cord injury, longer compression time can lead to more significant changes in the latency and amplitude of SEP, with the change in the amplitude more significant than that in the latency.
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Inulin has been used as a prebiotic to alleviate glucose and lipid metabolism disorders in mice and humans by modulating the gut microbiota. However, the mechanism underlying the alleviation of metabolic disorders by inulin through interactions between the gut microbiota and host cells is unclear. We use ob/ob mice as a model to study the effect of inulin on the cecal microbiota by 16S rRNA gene amplicon sequencing and its interaction with host cells by transcriptomics. The inulin-supplemented diet improved glucose and lipid metabolism disorder parameters in ob/ob mice, alleviating fat accumulation and glucose intolerance. The α diversity of gut microbial community of ob/ob mice was reduced after inulin treatment, while the β diversity tended to return to the level of wild type mice. Interestingly, Prevotellaceae UCG 001 (family Prevotellaceae) was obviously enriched after inulin treatment. A comparative analysis of the gene expression profile showed that the cecal transcriptome was changed in leptin gene deficiency mice, whereas the inulin-supplemented diet partially reversed the changes in leptin gene-related signaling pathways, especially AMPK signaling pathway, where the levels of gene expression became comparable to those in wild type mice. Further analysis indicated that Prevotellaceae UCG 001 was positively correlated with the AMPK signaling pathway, which was negatively correlated with markers of glycolipid metabolism disorders. Our results suggest that the inulin-supplemented diet alleviates glucose and lipid metabolism disorders by partially restoring leptin related pathways mediated by gut microbiota.
Subject(s)
Animals , Male , Mice , AMP-Activated Protein Kinases , Metabolism , Cecum , Metabolism , Microbiology , Gastrointestinal Microbiome , Inulin , Therapeutic Uses , Leptin , Genetics , Metabolic Diseases , Drug Therapy , Metabolism , Microbiology , Mice, Obese , Prebiotics , Signal Transduction , TranscriptomeABSTRACT
Objective To explore the efficacy of peroneus brevis tendon transfer and peroneal tendofascial flap for repairing achilles tendon and overlying skin defect.Methods From April,2004 to May,2015,5 cases of achilles tendon and overlying skin defect were treated with peroneus brevis tendon transfer and peroneal tendofascial flap.In these cases,the length of the achilles tendon defect was from 3.0 cm to 8.0 cm,and the size of skin defect ranged from 2.0 cm × 3.0 cm to 3.0 cm × 5.0 cm.Using peroneus brevis tendon to reconstruct achilles tendon defect,and covered with peroneal tendofascial flap,and then skin graft.One of them combined with the gastrocnemius tendon V-Y advancement to promote the reconstruction of achilles tendon longer defect.The patients were followed-up regularly,and evaluated by the American Society of Ankle Arrhythmia (AOFAS) ankle-hindfoot scoring system.ResultsAll operations were successful and the grafted skin survived.All cases were followed-up from 6 months to 10 years with an average of 5.2 years.At the time of last followed-up,all wounds healed well without re-infection and ulceration.Not foot varus deformity and the strongth was back to the level before the injury.AOFAS ankle-hindfoot score was increased from (50.44 ± 12.05)(preoperative) to (90.02 ± 6.55)(the last follow-up) (P＜0.05).Conclusion There were some advantages in the method of the treatment of achilles tendon and overlying skin defect by using peroneus brevis tendon transfer and peroneal tendofascial flap,such as easy to cut,small damage to the donor area,and no significant deformity to the receptor area,etc.It is a good way to repair achilles tendon and overlying skin defects.
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Objective To develop a modified method of adherent culturing NSCs to replace the traditional suspension culturing method.Methods Neural stem cells(NSCs)were isolated from brain of fetal mouse at 15.5-day fetal age.In culture bottle which had been pre-coated with poly-L-ornithine hydrochloride/fibronectin(PO/FN),the cells were adherently cultured and passaged.The cell morphology was observed under inverted microscope.NSCs and their differentiated cells were identified by immunofluorescence.Results NSCs obtained in this study were successfully adherently cultured and expressed specific markers.Conclusion NSCs are successfully adherently cultured in this study.As compared with the traditional suspension culturing method,adherent culturing is simpler and has lower wastage of passage.It is easy to observe cell morphology and differentiation by this method.It is also helpful to culturing and storage of NSCs.
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Objective To explore the expression profile of Wnt4 in rat kidney during renal development and its effect on renal development. Methods Rats with embryonic age of 18 days (E 18 d) , 20 days (E 20 d) as well as postnatal age of 0 day (P 0 d), 1 day (P 1 d), 3 days (P 3 d), 5 days (P 5 d) and 7 days (P 7 d) were selected. Expression levels of Wnt4 in rat kidney during renal development were quantified by immunohistochemistry and Western blot in all time points. Results Immuno?histochemistry analysis showed that during E 18 d to P 7 d, Wnt4 mainly expressed in proximal tubules, ureteric bud, comma shaped bodies and S shaped bodies of nephrogenic zone;the expression in the distal tubule was weak;the expression in renal corpuscle decreased with time;Western blot analysis showed that the expression of Wnt4 in rat kidney began to decrease from E 18 d and reached bottom at P 1 d then rise again until P 7 d when it dropped again. Conclusion During renal development, Wnt4 proteins were involved in the development of the nephrogenic zone through regulating canonical Wnt/β-catenin signaling pathway, and was involved in extension of proximal tubules by inducing the non canonical Wnt/PCP signaling pathway. Expression of Wnt4 protein in rat kidney was closely related to nephron formation and development of proximal tubules.
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Objective Getting the robust exogenous gene expression vector under the control of porcine insulin promoter, and to lay the foundation for pancreaticβ-cells specific transgene expressing pigs.Method Using porcine insu-lin promoter ( PIP, 1500 bp of the 5′UTR from the porcine INS gene including the first exon and the first intron) to con-struct expression vector, the HindIII restriction site which connected the sequences of PIP and EGFP was designed before ATG, named PIP-HindIII-EGFP.Considering that the different location of restriction site may affect the expression efficien-cy of the transgene, we optimized the expression vector.Firstly the HindIII restriction site was deleted to realize the seam-less connection of PIP and EGFP,the vector was named PIP-EGFP.Also we mutated the 3′intron splicing acceptor site( SA) of the first intron into HindIII restriction site, named as PIP-SA( M)-EGFP.Three different EGFP expression vectors were respectively transfected MIN-6 mouse pancreatic β-cells, pig ear fibroblasts and kidney cells.The transfected cells were cultured for 48 h and harvested for RT-PCR, flow cytometry and Western blot analysis, to analyze and compare the expres-sion efficiency of vectors.Results After transfection,green fluorescence was observed only in MIN-6 mouse pancreaticβ-cells.RT-PCR analysis and product sequencing showed that the three expression vectors did have different stability with in-tron splicing.The PIP-HindIII-EGFP construct and PIP-EGFP vector produced two kinds of mRNA with the first intron spliced and no spliced, indicating the instability of intron splicing.Mutation of the PIP splice site would cause the first in-tron not spliced, while flow cytometry and Western blot displayed that the mutation induced a most efficient expression of the downstream gene.Conclusions A robust and specific β-cells expression vector has been successfully generated by mutating the intron splicing acceptor site of the porcine insulin promoter.It provides the foundation for preparation of pigs with pancreaticβ-cells specifically expressing the transgene.
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Objective To observe the morphological changes of rats' pancreas and nitric oxide (NO) and endothelin(ET) in the blood serum in rats after exposure to different pulses of high power pulse microwave (HPPMW).Methods SD-rats were irradiated with 104,105 and 4 × 105 pulses of HPPMW,respectively.After gloss observation,the histopathological changes of pancreas were observed through biological microscope and electroscope.The changes of amylase,nitric oxide and endothelin in blood serum were detected by biochemical and radio-immunological methods. Results Compared with the blank control,no apparent abnormality could be observed in the pancreas of all groups.The dilatation of capillary could be observed in each experimental group by microscope.The ultrastructure changes of pancreas were most serious in 4 × 105 pulse group,especially at 24 and 48 h after irradiation.Compared with the control group,the levels of serum amylase were decreased (F =12.58,11.73,P ＜ 0.05),while ET were increased (F =4.50,4.49,P ＜0.05) at 24 and 48 h after irradiation.The levels of NO in serum were increased ( F =17.51,41.72,19.98,32.64,P ＜ 0.05 ) at each time-point.The level of NO went up with the increase of pulses.Conclusions HPPMW has damage effects on the pancreas in rats.The pulses with the pancreas can lead to severity of the damage. The mechanism of HPPMW may be involved in the enhancement of ET and NO in serum.
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Objective:To explore the mRNA expression of INSL3 gene in perinatal mice Leydig cells exposed to diethylstilbestrol(DES) in vivo and in vitro.Methods:The Leydig cells were isolated from fetal mouse testis and were cultured in DMEM/F12 medium and identified by 3 beta-hydroxysteroid dehydrogenase(3?-HSD).Reverse transcriptase-polymerase chain reaction(RT-PCR)and in situ hybridization were used for examination of the expression levels of INSL3 mRNA in primarily cultured Leydig cells incubated with DES(at the dose of 1?g/L,5?g/L and 25?g/L,respectively).Pregnant mice were exposed to DES at the dose of 1,10 or 100?g/kg body weight per day by gavage from gestation day(GD)12 to postnatal day(PND)3.The expression of INSL3 gene in neonatal mouse testis was investigated by RT-PCR.Results:Histological alterations of primarily cultured Leydig cells and neonatal mouse testis exposed to DES were observed.The expression level of INSL3 mRNA in DES treated groups including the primarily cultured Leydig cells and male mouse offspring’s testis were lower than those of the control groups.Conclusion:DES can downregulate the expression level of INSL3 mRNA in Leydig cells in vitro and in vivo.It may be a possible mechanism that DES interferes with gubernaculum development and cause cryptorchidism.