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 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.

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.

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.

Objective To investigate the expression and clinical pathological significance of PIWI-interacting RNA-47851 (piR-47851) in gastric adenocarcinoma and its influence on proliferation. Methods The expression of piR-47851 was detected in 79 gastric adenocarcinoma tissues by real time fluorescence quantitative polymerase chain reaction (qRT-PCR), and the correlation of piR-47851 expression level and clinical features with survival and prognosis were analyzed. The effect of piR-47851 on proliferation activity of gastric cancer cells was observed by cell proliferation experiments. Informatics websites were used to predict the downstream target genes of piR-47851. The wild-type and mutant plasmids for the 3'untranslated region (UTR) of MAPK1 gene were established, and a dual luciferase reporting system was used to verify that piR-47851 binded to the 3'UTR of MAPK1 gene, thereby inhibiting MAPK1 protein synthesis. The effect of overexpression/silencing of piR-47851 on MAPK1 expression level was examined through qRT-PCR experiment. Rescue experiment was used to confirm the direct regulatory effect of piR-47851 on MAPK1 and explore the effect of piR-47851/MAPK1 on the proliferation of gastric cancer cells. The effect of reduced piR-47851 expression on the volume and weight of transplanted tumors was observed in nude mice by xenograft tumor experiments; the effect of reduced piR-47851 expression on the cell proliferation activity of xenograft tumors was observed by Ki-67 staining. Results The qRT-PCR experiment result showed that the expression level of piR-47851 in 79 gastric adenocarcinoma tissues was significantly higher than that in normal gastric tissues adjacent to the cancer (P < 0.05). The expression level of piR-47851 was significantly correlated with tumor size, degree of differentiation, and survival prognosis (P < 0.05). Bioinformatics suggested that there was a complementary binding site between piR-47851 and MAPK1 gene 3'UTR. The dual luciferase reporter gene experiment showed that piR-47851 was able to significantly inhibit fluorescence enzyme activity in the MAPK1 wild-type transfection group, while this inhibitory effect was not observed in the MAPK1 mutant group. The CCK-8 proliferation activity experiment showed that overexpression of piR-47851 was able to significantly increase the proliferation activity of gastric cancer cells; after reducing piR-47851, the proliferation activity of cells decreased significantly (P < 0.05). Rescue experiment indicated that after changing the expression of piR-47851 and (or) MAPK1 and then detecting the expression level of MAPK1 by qRT-PCR, the MAPK1 could be regulated by piR-47851. Overexpression of MAPK1 could functionally reduce the proliferation promoting effect of piR-47851 on gastric cancer cells, while reducing MAPK1 expression could further enhance the proliferation activity of gastric cancer cells. In xenograft tumor experiments, reducing the expression of piR-47851 significantly resulted in smaller tumor growth volume and weight (P < 0.05). The proliferation index Ki-67 staining showed that the number of proliferative active cells in the reduced piR-47851 expression group was significantly lower than that in the control group (P < 0.05), which further validated the promotional effect of piR-47851 on cell proliferation. Conclusion The expression level of piR-47851 is increased in gastric adenocarcinoma tissues, which is closely related with tumor size and survival prognosis. The piR-47851 downregulates MAPK1 protein expression by binding to the 3'UTR of MAPK1, thereby promoting the proliferation of gastric cancer cells.

Objective To investigate the role of hsa_circ_0013058 in esophageal squamous cell carcinoma (ESCC) and the mechanism in promoting the invasion and migration of ESCC cells. Methods ESCC tissues and adjacent normal tissues of 75 patients were collected. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and RNA in situ hybridization (RISH) were used to detect the expression of hsa_circ_0013058, and the relationship between hsa_circ_0013058 and clinicopathological data of ESCC patients was analyzed. The invasion and migration ability of ESCC cells were detected by scratch test and Transwell test. The target gene of hsa_circ_0013058 was predicted by bioinformatics. The regulation of hsa_circ_0013058 on downstream genes and its effect on invasion and migration of ESCC cells were detected by Rescue experiment. Results The results of qRT-PCR showed that the expression level of hsa_circ_0013058 in the ESCC tissues was significantly higher than that in the adjacent normal tissues (t=5.078, P < 0.05). The expression level of hsa_circ_0013058 in KYSE70 was significantly higher than that in human normal esophageal epithelial cells HTT-1A (P < 0.05). At the RNA level, hsa_circ_0013058 was negatively correlated with miR-548p (r=-0.254 2, P < 0.05). The positive expression rate of hsa_circ_0013058 RNA in the ESCC tissues was 72.00%, which was significantly higher than 17.33% in the adjacent normal tissues (χ²=6.862, P < 0.05). The positive expression of hsa_circ_0013058 was closely related to the depth of tumor invasion, differentiation degree and lymph node metastasis (P < 0.05). Scratch test results showed that overexpression of hsa_circ_0013058 enhanced the migration ability of KYSE70 cells (P < 0.05); knockdown of hsa_circ_0013058 expression decreased the migration ability of KYSE70 cells (P < 0.05). After overexpression of hsa_circ_0013058, the number of invasion and migration cells of ESCC increased, and after knockdown of hsa_circ_0013058 expression, the number of invasion and migration cells decreased (P < 0.05). The qRT-PCR confirmed that hsa_circ_0013058 could regulate the expression of microRNA-548p (miR-548p). Dual luciferase reporter gene assay showed that miR-548p was specifically bound to LPAR3, which was the direct target gene of miR-548p. Rescue experiment results indicated that hsa_circ_0013058 regulated the invasion and migration of ESCC cells through the miR-548p/LPAR3 signaling axis. Conclusion In ESCC, hsa_circ_0013058 inhibits the expression of miR-548p, thereby activating the LPAR3 signaling pathway and promoting the invasion and migration of ESCC cells.

Objective To investigate the expression level of piR-35296 in clear cell renal cell carcinoma (ccRCC) and its pathological significance, as well as its effect on ccRCC cell proliferation. Methods Surgical resection specimens and clinicopathological data from 67 ccRCC patients were collected. Differential expression profiles of piRNAs in ccRCC were detected using piRNA microarray analysis, and validated by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The effect of piR-35296 on the proliferative activity of ccRCC cells was examined by CCK-8 assay. Bioinformatic analysis predicted TAB2 as a potential downstream target gene of piR-35296; qRT-PCR was performed to detect theinfluence of piR-35296 overexpression or knockdown on TAB2 expression levels. Rescue experiments were conducted to verify the regulation of TAB2 by piR-35296 and its impact on ccRCC cell proliferation. The effect of piR-35296 knockdown on the growth of subcutaneous tumors in experimental nude mice was analyzed, and Ki-67 immunohistochemical staining was used to validate the influence of piR-35296 on ccRCC cell proliferation. Results Both piRNA expression profiling and qRT-PCR revealed an elevated expression level of piR-35296 in ccRCC (P < 0.05). The expression of piR-35296 was closely associated with tumor size and differentiation degree in ccRCC patients (χ²=3.984, 3.974, P < 0.05). CCK-8 proliferation assays demonstrated that piR-35296 overexpression enhanced the proliferative activity of ccRCC cells, whereas its downregulation inhibited cell proliferation (P < 0.05). qRT-PCR experiments showed that TAB2 expression was upregulated after piR-35296 overexpression and downregulated upon piR-35296 knockdown (P < 0.05). Rescue experiments indicated that piR-35296 promoted TAB2 mRNA and its protein expression in ccRCC cells, thereby stimulating cell proliferation; TAB2 downregulation inhibited ccRCC cell proliferation. In experimental nude mice, piR-35296 knockdown reduced the mass andvolume of subcutaneous tumors (P < 0.05). Immunohistochemical staining for Ki-67 revealed a decreased percentage of Ki-67-positive cells after piR-35296 downregulation, suggesting reduced cell proliferation capacity (P < 0.05). Conclusion The piR-35296 serves as a novel oncogenic growth promoter in ccRCC. By elevating TAB2 expression, piR-35296 promotes the proliferative growth of ccRCC cells.