High content screening(HCS) analysis is an advanced technique in the era of systematic biology,which can comprehensively reflect the changes of cell with live,dynamic,and multiparametric characters.It is a good tool to study the complex ingredients and mechanism of traditional Chinese medicine(TCM).Application of the technique would contribute to the discovery of effective ingredients,elucidation of mechanism,and development of TCM theory.

Objective:To determine the molecular role of DNA methyltransferase (DNMT) in kidney tumorigenesis. Methods:Tissue samples consisted of 15 cancer tissues and 15 matched adjacent tissues from clear cell renal cell carcinoma (ccRCC) patients who had undergone nephrectomy in 2012 at the First Affiliated Hospital of Baotou Medical College, Science and Technology University of Inner Mongolia were collected. Real-time PCR, Western blot, combined bisulfite restriction analysis (COBRA) and methylation specific PCR were used in this study. Real-time PCR was used to examine the mRNA expression levels of DNMT. The global methylation level, DNA methylation level, and the expression of the antioncogene RASSF1A in ccRCC tissues were concurrently detected. Results:Both mRNA and protein levels of DNMT3B4, a splice variant of DNMT3B, were elevated in renal cell carcinoma tissue compared with those in con-troll tissue. Additionally, Alu was hypomethylated in ccRCC tissue (0.106±0.04) compared with control tissue (0.115±0.03) (P<0.05). Fur-thermore, the methylation of the promoter for RASSF1A, a tumor-suppressor gene, moderately increased in renal cell carcinoma tis-sue. By contrast, RASF1A expression decreased. Conclusion:DNMT3B4 overexpression may play an important role in human kidney tu-morigenesis via chromosomal instability and the decreased expression of RASSF1A.

Objective To explore effect of increasing cerebrospinal fluid (CSF) leptin level by administration from the foramen magnum to the cerebellum medulla oblongata pool in osteoporosis in rabbit model established by ovariectomy (OVX) and methylprednisolones.Methods According to certain inclusion and exclusion criteria,30 adult rabbits which were healthy 5-month-old female New Zealand white rabbits with the same batch and weight were divided into 5 groups (n=6) with random number table,including control group,normal saline group,Low dose group,middle dose group and high dose group.The animal model of osteoporosis (OP) was established 8 weeks after ovariectomy (bilateral ovarian resection) and intramuscular injection of methylprednisolone daily for 8 consecutive weeks in all groups.The control group were only simulated puncture and didn't inject any drugs or normal saline,the normal saline group were intracerebroventricular (ICV) injection with NS and Low,Middle and High dose group injected with low,middle and high dose of leptin respectively at 0,3,7 days after OVX.Before (0 week) and 4,8 weeks after the operation,the measure of dual energy X-ray absorptiometry was done on all the rabbits femur and bone mineral density (BMD) was delivered.Before (0 week) and 4,8 weeks after the first administration,leptin in cerebrospinal fluid (CSF) and serum growth hormone (GH),insulin growth factor 1 (IGF-1) and osteocalcin (OT/BGP) were tested with Enzyme linked immunosorbent assay and serum calcium,phosphorus and alkaline phosphatase (ALP) with automatic biochemical analyzer (ACA).The rabbits were sacrificed and the thalamus and femoral neck were harvested to test the expression of leptin receptor (LEPR) in thalamus and bone morphogenetic protein-2 (BMP-2) and type Ⅰ collagen (Col Ⅰ) at 8 weeks after the first administration.Results Compared with the control group and NS group,the cerebrospinal fluid (CSF) leptin concentration of leptin administration groups kept in a higher level significantly at 4 and 8 weeks after leptin administration,it was positively correlated with the concentration of administration.After administration 4 and 8 weeks,the bone mineral density (BMD) of leptin administration groups were not significantly lower than that of non-administration groups,and the high dose group was unchanged.The serum osteocalcin/bone glad protein (OT/BGP) concentration of leptin administration groups decreased more significantly compared with the non-administration groups at 4 weeks after leptin administration;and it rebounded more significantly than the non-administration groups and low dose group rebounded more significantly than other non-administration groups at 8 weeks.The concentration of serum growth hormone (GH) of 5 groups showed a downward trend at 4 and 8 weeks,the serum GH concentration of leptin administration groups decreased more significantly than the non-administration groups.The concentration of serum insulin-like growth IGF-1 of 5 groups were decreased significantly at 4 weeks,and the serum IGF-1 concentration of control and low dose groups were significantly higher than other 3 groups at 8weeks.Compared with the non-administration groups,the concentration of serum Calcium of leptin administration groups were increased more obvious,and it was positively correlated with the concentration of administration.Serum phosphorus of 5 groups were decreased at 4 weeks and elevated at 8 weeks;there was no difference among the groups.Serum ALP showed a continuous downward trend,and the middle dose group decreased significantly compared with other groups at 4 and 8 weeks.RTPCR results showed that the BMP-2 and Col Ⅰ mRNA expression increased and positively correlated with leptin administration concentration at 8weeks.Conclusion The increase of cerebrospinal fluid leptin level can promote the bone metabolism,reduce bone loss and precaution the occurrence of osteoporosis in rabbit model caused by OVX and methylprednisolone,and it was positively correlated with the concentration of administration.That provides a new method for the prevention and treatment of osteoporosis in rabbit model.

To investigate the effects and characteristics of Fuzheng Huayu recipe, a compound Chinese herbal medicine, and its decomposed therapies against hepatocyte apoptosis in mice with hepatic injury.

Objective:Ciliary neurotrophic factor (CNTF) plays important roles in the maintenance and survival of motor neurons. This study attempted to explore the expression and distribution of CNTF mRNA and its protein in the ambiguous nucleus(Amb) motoneuron in order to clarify its functional state after long term laryngeal denervation. Methods: The recurrent laryngeal nerves were obtained from dogs. Brain stems were removed and sectioned for histochemistry, immunohistochemistry and in situ hybridization of CNTF. Amb motoneurons were identified by Nissl staining. The count and intensity of positive reactive motoneurons were measured by computer image processing system. Results: Transection of the laryngeal nerve led to a very marked reduction in the count and intensity of CNTF mRNA positive reactive motoneurons, and reached minimal levels at week 3. CNTF immunoreactivity increased rapidly and reached maximal levels also at week 3. At week 4, a significant increase in CNTF mRNA expression and decrease in CNTF immunoreactivity were observed. At week 6, both CNTF mRNA and its protein expression were significantly less than those of unlesioned contraletaral sides. Although a difference between week 6 and 12 was observed, the motoneurons were generally stable in the expression level of CNTF mRNA and its protein, and in the size and count after 12 weeks, with 78%, 84.4%, 80.9% and 83.7% respectively as compared with the unlesioned contralateral Amb. Conclusion: The results indicate that although degenerating changes occurre in the Amb motoneurons after long term laryngeal denervation, the ciliary neurotrophic factors activity of the lesioned motoneurons is still maintained at a certain level. [

OBJECTIVETo observe the effects of cross electro-nape-acupuncture on reflex remodeling of airway protective reflex cough in patients with tracheotomy after cerebral hemorrhage.

METHODSWith the method of completely random design, according to treatment order, 60 patients who received tracheotomy after cerebral hemorrhage accompanied with cough reflex difficulty were randomly divided into a cross electro-nape-acupuncture group and an acupuncture group, 30 cases in each group. Both groups were treated with basic treatment, including anti-inflammation, eliminating phlegm, improving cerebral metabolism and so on. The acupuncture group was treated with acupuncture at Yifeng (TE 17), Fengchi (GB 20), Lianquan (CV 23), Baihui (GV 20), Touwei (ST 8), Dicang (ST 4) through Jiache (ST 6), Hegu (LI 4), Quchi (LI 11), and motor area on the affected side, and the needles were retained for 30 min. Based on the treatment of acupuncture group, the cross electro-nape-acupuncture group was additionally treated with cross electro-nape-acupuncture (continuous wave) for 30 min per treatment. The treatment was both given twice a day from Monday to Friday and once a day on Saturday and Sun day for 4 weeks. Tracheostomy cough reflex grading score (TCRGS) and clinical pulmonary infection score (CPIS) were observed before and after treatment in the two groups, and the clinical efficacy of two groups was evaluated.

RESULTSCompared before the treatment, TCRGS and CPIS were both reduced in two groups (both P < 0.01); after treatment, there were significant differences of TCRGS and CPIS between two groups (both P < 0.01), indicating cross electro-nape-acupuncture group was superior to acupuncture group. Regarding the effects of cough reflex remodeling, the cured and markedly effective rate was 96.7% (29/30) in the cross electro-nape-acupuncture group, which was significantly different from 55.2% (16/29) in the acupuncture group (P < 0.01).

CONCLUSIONCross electro-nape-acupuncture could effectively improve the remodeling of cough reflex and promote the recovery of lung infection in patients with tracheotomy after cerebral hemorrhage, leading to an increased quality of life.

Acupuncture Points ; Aged ; Cerebral Hemorrhage ; physiopathology ; surgery ; therapy ; Cough ; physiopathology ; Electroacupuncture ; Female ; Humans ; Male ; Middle Aged ; Reflex ; Tracheotomy

Objective:To explore the expression and distribution of ciliary neurotrophic factor (CNTF) in laryngeal nerve degeneration and regeneration. Methods:Transection of the recurrent laryngeal nerves in 8 dogs and suture following transection in 12 cases were performed. Both proximal and distal ends of transected or sutured region were sectioned at various survival times for CNTF immunohistochemistry and CNTF mRNA in situ hybridization. The area and intensity of reactive product were measured by computer image processing system. Results:After nerve transection, reactive product of CNTF mRNA and its protein reduced rapidly in distal stumps, after neurorrhaphy, they were observed in thin Schwann cell processes ensheathing axons and not found in the proliferating Schwann cells which didn′t ensheathe axons. CNTF immunoreactivity was also detected in the regenerated nerve axons. CNTF expression increased with survival time, but even at the longest survival time, it was still significantly less than that in intact nerve. The same change was observed in a short segment proximal to the transected or sutured region. Conclusion:CNTF expression is in the down regulation and is collected with Schwann cell axon in peripheral nerve degeneration and regeneration. The changed distribution of CNFT might provide a supportive environment for axonal regeneration.

OBJECTIVETo investigate the effects of Fuzheng Huayu recipe and Huangqi tang on DMN-induced experimental liver cirrhosis in rats and explore the therapeutic characteristics of Buxu herbals on liver cirrhosis.

METHODLiver cirrhosis in rats was induced by intraperitoneally injection of DMN for 4 weeks. Cirrhotic rats were randomly divided into 4 groups: model group, and Fuzheng Huayu recipe group, Huangqi tang group, Fuzheng Huayu recipe combined with Huangqi Tang group. The rats in treatment groups were orally administered with Fuzheng Huayu recipe, Huangqi tang, Fuzheng Huayu recipe combined with Huangqi tang (1:1), respectively. Normal and model control rats were given the equivalent normal saline. The body weight, liver weight and spleen weight were observed when rats were sacrificed. Liver histology was examined by HE staining and Sirius red staining. The liver function parameters including ALT, T. Bil and Alb were determined. The SOD activity and MDA content in liver tissues were also measured. Hepatic hydroxyproline (Hyp) content was determined by Jamall's method. The expression of alpha-SMA was determined by both immunohistochemistry staining and western blot method.

RESULTCompared with normal rats, the serum ALT and T. Bil levels in model rats increased obviously, by contrast, the serum Alb level decreased. There was a significant decline of SOD activity in model rat liver tissue, while the content of MDA and Hyp increased remarkably. A severe deterioration of liver architecture, infiltration of inflammatory cells and deposition of collagen were observed in model rat liver tissue. The expression of alpha-SMA also increased significantly. Compared with model rats, the liver function, lipid peroxidation parameters, Hyp content and liver histology were all improved in the 3 treatment groups. The combined group is better than any single-use group in decreasing collagen deposition and expression of alpha-SMA.

CONCLUSIONFuzheng Huayu recipe, Huangqi tang, Fuzheng Huayu recipe combined with Huangqi tang can attenuate liver fibrosis in DMN induced rats. Fuzheng Huayu recipe combined with Huangqi tang is better than that using alone in decreasing collagen deposition. The mechanism is partially due to the better effect of Fuzheng Huayu recipe combined with Huangqi tang on inhibiting activated HSC.

Animals ; Body Weight ; drug effects ; Dimethylnitrosamine ; adverse effects ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Liver ; drug effects ; enzymology ; Liver Cirrhosis, Experimental ; chemically induced ; drug therapy ; enzymology ; physiopathology ; Organ Size ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism

ObjectiveTo investigate the clinical promoting effect of crossing nape electroacupuncture on the recovery of swallowing function and recovery from pulmonary infection in post-cerebral infarction patients with tracheotomy.MethodSixty post-cerebral infarction patients with cough reflex disorder and swallowing dysfunction associated with pulmonary infection receiving tracheotomy and tracheal intubation were subjects. They were allocated, using a random number table, to three groups, 20 cases each. In each group, the patients were enrolled in order of visits. The three groups were given the same basic treatment for fighting inflammation, resolving phlegm and improving blood supply. The crossing nape electroacupuncture group received bilateral points Fengchi (GB20), Yifeng (TE17), Dicang (ST4)-to-Jiache (ST6) and Lianquan (CV23) acupuncture with electrodes connected by left-right crossing. The acupuncture group received bilateral points Fengchi, Yifeng, Dicang-to-Jiache and Lianquan acupuncture without electrodes connected. The control group received basic treatment with Western drugs without acupuncture therapy. Observations were carried out using the Kubota’s water drinking test, the Toshima Ichiro Swallowing Assessment and the Clinical Pulmonary Infection Score. The clinical therapeutic effects were evaluated in the three groups.ResultThe therapeutic effects evaluated using the Kubota’s water drinking test and the Toshima Ichiro Swallowing Assessment were better in the crossing nape electroacupuncture group than in the acupuncture group and better in the acupuncture group than in the control group (P<0.05). The score of the Clinical Pulmonary Infection Score decreased in all the three groups. The promoting effect on recovery from pulmonary infection was marked in the crossing nape electroacupuncture group (P<0.01).ConclusionCrossing nape electroacupuncture has a marked improving effect on dysphagia in post-cerebral infarction patients with tracheotomy and tracheal intubation. It can promote recovery from pulmonary infection in post-cerebral infarction patients with cough reflex disorder receiving tracheotomy and tracheal intubation.