Objective] To analyze the medication experience of Zhong Yi-tang for insomnia by using TCM inheritance support system 1.1ver.[Methods] The prescriptions of Zhong Yi-tang for insomnia were collected and input the data into TCM inheritance support system .The frequency and association rules of drugs were calculated by using data mining methods such as apfiofi algorithm,complex system entropy cluster and unsupervised hierarchical cluster,and medication rule of Zhong Yi-tang for epigastric pain was analyzed.[Results] Based on the analysis of 354 prescriptions, the frequency of each herb and association rules among the herbs were computed. Spine date seed, tuber fleeceflower stem, paeony, red sage root, boxthorn and Chinese Date were highly frequently found, 18 pairs of Chinese medicines based on these and 5 core combinations such as ganmai-dazao decoction were mined out . [Conclusion] Zhong Yi-tang usually used ganmai-dazao decoction, spine date seed, tuber fleeceflower stem and red sage root to cure insomnia. The sweet and moistening Chinese medcine were used to strengthen-spleen-stomach, benefiting ying-blood, and nourishing the heart-blood. Data mining method can be used to analyze old TCM doctors’clinical experience.

ObjectiveTo investigate the effect and significance of cyclooxygenase-2 (COX-2) inhibitors on the expression of the Acsl gene family in the ileum of rats with nonalcoholic fatty liver disease (NAFLD). MethodsA total of 45 Sprague-Dawley rats were randomly divided into normal control group (15 rats given normal diet), NAFLD model group (15 rats given high-fat diet), and nimesulide group (15 rats given high-fat diet and nimesulide). All rats were sacrificed after 12 weeks of feeding, and then blood samples were collected from the inferior vena cava to measure total cholesterol (TC) and triglyceride (TG). HE staining and oil red O staining were performed for the liver to evaluate the degree of hepatic steatosis in each group, and quantitative real-time PCR was used to measure the mRNA expression of the Acsl family genes in the ileum. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the normal control group, the NAFLD model group had significant increases in serum TC and TG and marked hepatic steatosis (all P＜0.05); compared with the NAFLD model group, the nimesulide group had significant reductions in serum TC and TG and degree of hepatic steatosis (all P＜0.05). Compared with the normal control group, the NAFLD model group had a significant increase in the expression of COX-2 in the ileum (P＜0.05), and compared with the NAFLD model group, the nimesulide group had a significant reduction in the expression of COX-2 in the ileum (P＜005). Compared with the normal control group, the NAFLD model group had significant increases in the mRNA expression of Acsl3 and Acsl5 in the ileum (both P＜0.05), and compared with the NAFLD model group, the nimesulide group had significant reductions in the mRNA expression of Acsl3 and Acsl5 (both P＜0.05). ConclusionThe COX-2 inhibitor nimesulide can regulate the expression of the Acsl gene family in the ileum of rats with NAFLD, suggesting that COX-2 inhibitors may inhibit the progression of NAFLD through the Acsl gene.

ObjectiveTo obtain HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect by transfecting HSC-T6 cells with CRISPR/Cas9 lentiviral vector, and to provide a good method for further functional research and new strategies for the clinical treatment of liver fibrosis. MethodsThe COX-2 gene-specific sgRNAs (COX-2-sgRNA-1, COX-2-sgRNA-2, COX-2-sgRNA-3) were designed, synthesized, and connected to the GV371 vector, and the recombinant plasmid and the packaging plasmid were transfected into 293T cells to form lentivirus particles; the fluorescence method was used to measure virus titer. The most appropriate amount of the virus was calculated based on MOI. Lenti-Cas9-puro was transfected into HSC-T6 cells, and HSC-T6-Cas9 cells were screened out by puromycin; Lenti-COX-2-sgRNA-EGFP was transfected into HSC-T6-Cas9 cells to obtain HSC-T6-COX-2-/- cells. Cruiser enzyme digestion and Western blot were used to verify gene knockout at the gene and protein levels. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsSequencing verified that the COX-2-sgRNA expression vector was constructed successfully. Recombinant expression plasmids and packaging plasmids were transfected into 293T cells to form lentivirus particles, and the fluorescence method showed a virus titer of ＞1×108. HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect were successfully constructed. The HSC-T6-Cas9 group had significantly higher relative mRNA expression of LV-Cas9-Puro than the CON group (541.93±105.76 vs 1.00±0.02, t=12.995, P＜0.01). Cruiser enzyme digestion and Western blot showed that the CRISPR/Cas9 lentivirus expression vectors played a role in the target, among which COX-2-sgRNA-2 knockout had the most significant effect, and this group had a significant reduction in the protein expression level of COX-2 compared with the CON group and the NC group (both P＜0.05), suggesting that COX-2-sgRNA was active. ConclusionA CRISPR/Cas9 lentivirus vector is successfully constructed for COX-2 target gene, and HSC-T6-COX-2-/- cells with stable COX-2 gene knockout are obtained.

OBJECTIVE： To prepare Baicalin-loaded Polyethylene glycol-derivatized phosphatidylethanolamine （BAI@PEG-PE） nanomicelles， and to characterize it and study its cytotoxicity. METHODS： BAI@PEG-PE nanomicelles were prepared by film hydration method and their appearance characteristics were observed. The particle size， polydispersity index， Zeta potential， drug-loading amount and encapsulation efficiency of the nanomicelles were detected. Drug release of BAI raw material and BAI@PEG-PE nanomicelles in pH 7.4 phosphate buffer were compared within 1-84 h. Using coumarin 6 as fluorescent probe， the distribution of PEG-PE nanomicelles in H9c2 cardiomyocytes were observed. H9c2 cardiomyocytes were divided into model group， BAI raw material group and BAI@PEG-PE nanomicelles group. After treated with serum-free DMEM medium containing no or corresponding drugs for 0.5 h， isoproterenol was used to induce cardiomyocyte apoptosis. Nuclear morphology， cell apoptosis rate and protein expression of Bcl-2 and Bax were compared with among 3 groups. RESULTS： Prepared BAI@PEG-PE nanomicelles were uniform globular shape. The particle size was （16.7±0.8） nm， PDI was 0.11±0.01 and Zeta-potential was （－18.4±0.6） mV； drug-loading amount was （7.84±0.65）％， encapsulation efficiency was （85.7±4.9）％ （n＝3）. Accumulative release rate was 76.5％ within 84 h. BAI raw material was released completely within 24 h. PEG-PE nanomicelles could strengthen the intake of coumarin 6 in H9c2 cardiomyocytes， mainly gathering around mitochondria. Compared with model group， the apoptosis morphology of cardiomyocytes were improved significantly in BAI raw material group and BAI@PEG-PE nanomicelles group； apoptosis rate was decreased significantly； protein expression of Bcl-2 was increased significantly； protein expression of Bax was decreased significantly with statistical significance （P＜0.05 or P＜0.01）. Above effects of BAI@PEG-PE nanomicelles group were more significant （P＜0.05 or P＜0.01）. CONCLUSIONS： BAI@PEG-PE nanomicelles are prepared successfully， and show significant sustained-release effect and myocardial targeting， and can prevent cardiomyocyte apoptosis.

OBJECTIVETo observe the clinical effects of Jiaji (EX-B2) needling for treatment of spastic cerebral palsy (SCP).

METHODSSixty-two SCP patients were randomized into two groups, the treatment group treated by needling, and the control group treated by sham-acupuncture, once every day for 60 times totally. The clinical effect was assessed with scoring by the modified Ashworth's scale (MAS), gross motor function measuring scale (GMFM) and WeeFIM at the end of treatment (T1), half-year (T2) and 1-year (T3) after treatment.

RESULTSClinical effectiveness assessment showed that MAS scores in the treatment group reduced significantly and steadily at all the time points (P < 0.01), while in the control group, it reduced significantly at T1 (P < 0.01), but raised again at T2 and T3 to higher than that in the treatment group (P < 0.05, P < 0.01), approaching the baseline level (P > 0.05). GMFM and WeeFIM scores increased in both groups at all the three assessing time points (P < 0.01), but the increments in the treatment group were higher than those in the control group respectively (P < 0.01, P < 0.05).

CONCLUSIONJiaji (EX-B2) needling could achieve good clinical therapeutic effects on SCP.

Acupuncture Points ; Acupuncture Therapy ; methods ; Cerebral Palsy ; therapy ; Child, Preschool ; Female ; Follow-Up Studies ; Humans ; Infant ; Male

Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, autoimmune, drug-induced, cholestatic and metabolic diseases. Fibrosis is driven by a dynamic process involving increased synthesis of matrix components and a failure of physiological mechanisms of matrix turnover. Activation of hepatic stellate cells (HSCs) remains a central event in fibrosis. HSCs are the main source of extracellular matrix (ECM). Transforming growth factor-beta (TGF-Β), which is the fibrogenic master cytokine, can induce the activation of HSCs to produce a large amount of ECM, and is capable of inducing apoptosis of liver cells. RNA interference (RNAi) is a novel gene disruption technology. Studies have shown that small interfering RNA (siRNA) targeting TGF-Β1 may inhibit the activation and proliferation of HSCs, suppress ECM synthesis and block liver fibrosis. TGF-Β1 siRNA-mediated gene silencing therapy provides a new avenue for liver fibrosis. This review summarizes recent progresses in research on HSCs, TGF-Β1 and TGF-Β1 siRNA in liver fibrosis.
Liver Cirrhosis ; therapy ; RNA, Small Interfering ; genetics ; Transforming Growth Factor beta1 ; genetics

OBJECTIVEThis clinical study was to improve the surgical treatment to craniomaxillofacial tissue defects.

METHODSSince 1997, eight cases with severe craniomaxillofacial defects were treated using free latissimus dorsi myocutaneous flaps. In the operation, nerve anastomosis was performed. Of the 8 cases, 7 were treated in one stage, 1 was treated in 3 steps. The craniomaxillofacial defects ranged from 10 cm x 8 cm to 30 cm x 12 cm. The flaps was 12 cm x 10 cm to 32 cm x 16 cm in size.

RESULTSPostoperative follow-up for 6 months to 4 years demonstrated satisfactory results in all the cases. There was neither necrosis nor ulcer after the operation. The sensation recovery of the flap was also satisfactory.

CONCLUSIONFree transfer of the latissimus dorsi myocutaneous flap is an ideal treatment to severe craniomaxillofacial defects as it possesses the advantages of reliable blood supply, ability against infections, large size, concealed donor site, and functional restoration of sensation and movement.

Adult ; Aged ; Craniofacial Abnormalities ; surgery ; Female ; Humans ; Male ; Maxillofacial Abnormalities ; surgery ; Middle Aged ; Muscles ; metabolism ; surgery ; Reconstructive Surgical Procedures ; methods ; Surgical Flaps ; Transplantation, Homologous ; Treatment Outcome

To investigate the inhibition of cyclosporin A (CsA) on neutrophil adhesion to human umbilical vein endothelial cells (HUVECs, ECV-304) induced by hypoxia/reoxygenation and further explore its mechanism, a 1 h hypoxia/4 h reoxygenation model was reproduced using ECV-304. The adhesion rate of neutrophils to ECV-304 was determined by measuring the activity of endogenous hexosaminidase. The expression of endothelial cell adhesion molecules of E-selectin and ICAM-1 was measured by flow cytometry. The expression of cyclophilin A (CyPA) and the activation of ERK1/2 was compared among experimental groups by Western blot. The content of reactive oxygen species (ROS) was measured by Fenton reaction. After being stimulated with 1 h hypoxia/4 h reoxygenation, ECV-304 showed an enhanced neutrophil adhensiveness in association with an increased surface expression of E-selectin and ICAM-1. In parallel, the content of ROS was also increased. These effects were significantly suppressed by the addition of CsA. Most importantly, the expression of CyPA was significantly increased following 1 h hypoxia/4 h reoxygenation, which was accompanied with an increased activation of ERK1/2. Treatment with CyPA inhibitor CsA and CyPA antisense oligonucleotides significantly inhibited the activation of ERK1/2 and decreased the adhesion of neutrophils to ECV-304. The specific ERK1/2 inhibitor PD98059 caused an inhibition of neutrophil adhesion to hypoxia/reoxygenation-stimulated ECV-304. Our data confirm that CsA inhibits neutrophil adhesion to hypoxia/reoxygenation stimulated ECV-304 by a mechanism involving inhibition of the signal transduction of ROS, CyPA and ERK1/2.
Cell Adhesion ; Cell Hypoxia ; Cells, Cultured ; Cyclophilins ; biosynthesis ; genetics ; Cyclosporine ; pharmacology ; Endothelium, Vascular ; cytology ; Humans ; Intercellular Adhesion Molecule-1 ; biosynthesis ; genetics ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Neutrophils ; cytology ; Reactive Oxygen Species ; metabolism ; Reperfusion Injury ; physiopathology ; Signal Transduction ; Umbilical Veins ; cytology

Adolescent ; Adult ; Ankle ; pathology ; Arthroscopy ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Recovery of Function ; Soft Tissue Injuries ; diagnostic imaging ; physiopathology ; surgery ; Tomography, X-Ray Computed

AIMTo study the formation and localization of ADM mRNA in lung tissues and investigate the effects of ADM on isolated tracheal strip contraction induced by histamine in asthmatic guinea pig.

METHODSThe guinea pigs (n = 22) were randomly divided into two groups of 11 each: asthmatic group and control group. The formation and localization of ADM mRMA were observed by in site hybridization. The effect of exogenous ADM on contractions of isolated tracheal strip of the asthmatic guinea pigs to histamine was examined.

RESULTSThere were strong positive expression for ADM mRNA in airway epithelial cells (AEC), smooth muscle cells (ASMC) in asthmatic group. The control group showed significantly decreased number of ADM mRNA positive cells in lung tissues. From 10(-11) mol/L to 10(-7) mol/L, ADM may cause concentration depend pentiation of the isolated tracheal strip contraction induced by histamine of asthmatic group which was higher significantly compared the control group (P < 0.05). 10(-8) mol/L ADM reached the maximal relaxation, with the increasing of ADM, neither asthmatic nor control group can increase the relaxation.

CONCLUSIONThere is ADM mRNA overproduction in AEC and ASMC and exogenous ADM may inhibit isolated tracheal strip contraction induced by histamine of asthmatic guinea pig, which may contribute to airway inflammation and hyperresponsiveness in asthma.

Adrenomedullin ; metabolism ; Airway Resistance ; Animals ; Asthma ; metabolism ; physiopathology ; Guinea Pigs ; In Vitro Techniques ; Lung ; metabolism ; Male ; Trachea ; physiopathology