Objective To investigate the effect of cinobufacini on inhibiting colorectal cancer metastasis by regula-ting the polarization of M2 macrophages.Methods THP-1 was induced into M0 type macrophages.The condi-tioned medium of HCT116 cells was collected to stimulate M0 type macrophages.The polarization of M2 type mac-rophages was observed by flow cytometry,real-time quantitative PCR and ELISA experiments.The conditioned me-dium of M0 type macrophages and HCT116-Mφ cells was collected to stimulate HCT116 cells.The ability of migra-tion and invasion was observed by wound healing assay and Transwell assay.The effect of cinobufacini on the via-bility of HCT116 cells was detected by CCK-8 assay.The conditioned medium of HCT116 and HCT116+cinobufa-cini was collected to stimulate M0 type macrophages.The polarization of M2 type macrophages was observed by flow cytometry,real-time quantitative PCR and ELISA experiments.The conditioned media of HCT116-Mφ cells and(HCT116+cinobufacini)-Mφ cells were collected to stimulate HCT116 cells.The changes of migration and inva-sion ability were observed by wound healing assay and Transwell assay.Results After stimulation of M0 type mac-rophages in HCT116 cell conditioned medium,the morphology of M0 macrophages turned into fusiform cells,the proportion of CD11b+CD206+cells increased,and the expression of M2 macrophage markers IL-10 and TGF-β in-creased.The migration and invasion ability of HCT116 cells were significantly enhanced after stimulation in the conditioned medium of HCT1 16-Mφ cells.After the addition of cinobufacini,not only the polarization proportion of M2 macrophages decreased,but also the metastatic effect mediated by M2 macrophages was inhibited.Conclusion HCT116 cells can induce the polarization of M2 macrophages,while cinobufacini can inhibit the tumor metastasis mediated by M2 macrophages by inhibiting the polarization of M2 macrophages.

OBJECTIVE To study the effects of the Mongolian medicine Sugemule-4 on the metabolism of insomnia rats， and to preliminarily explore its possible mechanisms for improving insomnia. METHODS The rat model of chronic stress insomnia was established by tail clipping stimulation and intraperitoneal injection of p-chlorophenyl alanine solution. Twenty-four male rats were randomly divided into the normal group， model group， diazepam group （positive control， 0.92 mg/kg）， and Sugemule-4 group （5.2 g/kg）， with 6 rats in each group. Since the 7th day of tail clipping stimulation， the Sugemule-4 group and diazepam group began to be intragastrically administered with relevant medicine； the normal group and model group were intragastrically administered with an equal volume of distilled water， once a day， for 14 consecutive days. The learning and memory abilities of rats were tested using a water maze experiment， and the non-invasive sleep activity monitoring system was used to monitor the 24- hour sleep time of rats. A metabolomics study was conducted on rat serum and hippocampal tissue by using ultra-high-performance liquid chromatography-tandem mass spectrometry. The multivariate statistical analysis method was adopted to analyze the differential metabolites in serum and hippocampal tissue of rats， and screen for differential metabolites and metabolic pathways among those groups. RESULTS Compared with the normal group， the escape latency of rats in the model group was significantly increased， the times of crossing platforms were significantly reduced， and the percentage of average 24-hour sleep time was significantly reduced （P＜0.05）. Compared with the model group， the levels of the above indicators were significantly reversed in the diazepam group and Sugemule-4 group （P＜0.05）. Metabolomics studies found that a total of 9 differential metabolites were identified in rat serum and hippocampal tissue， including 5-hydroxyindoleacetic acid， canine urate， canine urinary quinolinic acid， 5-hydroxytryptamine， phenol sulfate， 1-carboxyethyltyrosine， 3-（4-hydroxyphenyl） lactate， N-acetyl tyrosine， tyrosine and phenol sulfate， mainly involving 2 metabolic pathways of tryptophan and tyrosine.CONCLUSIONS Sugemule-4 can improve the sleep time and behavioral performance of insomnia rats， and its mechanism may be associated with affecting amino acid metabolic pathways such as tryptophan and tyrosine.

Objective : To study the effect of bufalin on the proliferation and apoptosis of human colorectal cancer cell line HCT116，and to explore the role of endoplasmic reticulum stress ( ERS) in this process． Methods : The effect of bufalin on the proliferation of HCT116 cells was determined by CCK-8 assay．After HCT116 cells were treated with different concentrations of bufalin for 48 hours，cell apoptosis was detected by Annexin V / PI assay， and the expression of apoptosis-related proteins Bax and Bcl-2 was detected by Western blot．At the same time，the expression of ERS-related proteins glucose regulated protein 78 ( GRP78) ，phosphorylated protein kinase R like endoplasmic reticulum kinase ( p-PERK) ，eukaryotic translation initiation factor 2 α ( eIF2 α) ，phosphorylated eukaryotic translation initiation factor 2 α (p-eIF2 α) and C / EBP homologous protein ( CHOP) was detected by Western blot．HCT116 cells were divided into control group，bufalin group and combination group (bufalin + 4-phenylbutyric acid) ，and the expression of apoptosis-related proteins Bax and Bcl-2 was observed by Western blot. Results: CCK-8 assay showed that bufalin could inhibit the proliferation of HCT116 cells．Apoptosis assay showed that bufalin could induce apoptosis of HCT116 cells．The results of Western blot showed that bufalin could up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2．It could also induce ERS and activate PERK / eIF2 α/ CHOP pathway．When bufalin combined with 4-phenylbutyric acid，the apoptosis-promoting effect of bufalin was inhibited． Conclusion Bufalin can effectively inhibit the prolif- erative activity and induce apoptosis of HCT116，which is achieved to some extent by activating ERS.

Few studies have described the key features and prognostic roles of lung microbiota in patients with severe community-acquired pneumonia (SCAP). We prospectively enrolled consecutive SCAP patients admitted to ICU. Bronchoscopy was performed at bedside within 48 h of ICU admission, and 16S rRNA gene sequencing was applied to the collected bronchoalveolar lavage fluid. The primary outcome was clinical improvements defined as a decrease of 2 categories and above on a 7-category ordinal scale within 14 days following bronchoscopy. Sixty-seven patients were included. Multivariable permutational multivariate analysis of variance found that positive bacteria lab test results had the strongest independent association with lung microbiota (R² = 0.033; P = 0.018), followed by acute kidney injury (AKI; R² = 0.032; P = 0.011) and plasma MIP-1β level (R² = 0.027; P = 0.044). Random forest identified that the families Prevotellaceae, Moraxellaceae, and Staphylococcaceae were the biomarkers related to the positive bacteria lab test results. Multivariable Cox regression showed that the increase in α-diversity and the abundance of the families Prevotellaceae and Actinomycetaceae were associated with clinical improvements. The positive bacteria lab test results, AKI, and plasma MIP-1β level were associated with patients' lung microbiota composition on ICU admission. The families Prevotellaceae and Actinomycetaceae on admission predicted clinical improvements.
Acute Kidney Injury/complications* ; Bacteria/classification* ; Chemokine CCL4/blood* ; Community-Acquired Infections/microbiology* ; Humans ; Lung ; Microbiota/genetics* ; Pneumonia, Bacterial/diagnosis* ; Prognosis ; RNA, Ribosomal, 16S/genetics*

Objective:To observe the curative effects of berberine in rats with high-fat diet induced non-alcoholic fatty liver and to further explore its possible mechanism.Methods:Twenty-six Sprague-Dawley rats (120-160 g) were randomly divided into 3 groups: control group ( n = 8), model group ( n = 10) and treatment group ( n = 8). Rats in the control group were fed with regular diet, and the model group and the treatment group were fed a high-fat diet. At the 12th week, two rats in the in the model group were sacrificed to verify whether model was successful established. Subsequently, treatment group rats were given a gavage of berberine at a dose of 150 mg·kg -1·d -1 for 4 weeks, and the control and the model group rats were given the same dose of normal saline. Rats were sacrificed at week 16th. HE staining was used to observe the changes in the intestinal mucosa of rats. Sudan black B staining was used to observe the fatty changes in liver. Immunohistochemical staining was used to observe the expression level of occludin protein in the intestinal epithelium. A real-time 16S rDNA PCR method was used to measure the number of escherichia coli, bacteroides and faecalibacterium prausnitzii in the feces of rats. Results:Model group had a higher serum levels of endotoxin (0.288 ± 0.045) and tumor necrosis factor (TNF)-α (1.07 ± 0.11) than the control group (0.192 ± 0.049, 0.94 ± 0.07) ( P < 0.05). Berberine intervention had significantly reduced endotoxin (0.213 ± 0.025) and TNF-α level (0.93 ± 0.07) ( P < 0.05). The expression level of occludin protein was significantly lower in the intestinal mucosa of model group than that of control group (0.166 ± 0.014), and berberine had promoted the expression of occludin protein in intestinal mucosa (0.055 ± 0.009), but the difference was not statistically significant ( P > 0.05). At the same time, compared with the model group (7.29 ± 0.47), the number of bacteroidetes in the control group (9.49 ± 0.59) was decreased, while the number of bacteroidetes in the treatment group was increased (9.77 ± 0.87). The number of escherichia coli (6.92 ± 0.77) and faecalibacterium prausnitzii (8.70 ± 0.62) in the model group were increased than control group (5.42 ± 0.63, 9.49 ± 0.59), while the number of escherichia coli (6.34 ± 0.71) and faecalibacterium prausnitzii (9.77 ± 0.87) ( P < 0.05) was reduced with the intervention of berberine. Conclusion:Berberine could effectively protect the intestinal barrier function in rats with NAFLD and the possible mechanism of action behind it may be the regulation of intestinal flora.

Objective: Alport syndrome was an inherited kidney disease caused by the mutation of COL4A3, COL4A4, or COL4A5. Whole-exome sequencing was used to detect the mutations on these genes for the molecular diagnosis of Alport syndrome. Methods: A 6-year-old girl found accidentally with microscopic hematuria at the age of 4. The clinical data and blood sample of the family including proband, parents, brothers, and sisters were collected. Whole exome sequencing was conducted using their genomic DNAs. Results: A novel heterozygous frameshift mutation c.1826delC (p.Pro609Glnfs*44) was found in the exon 25 of the COL4A4 (NM_000092) in the proband, the father, and the sister, showing an autosomal dominant inheritance pattern of Alport syndrome. This mutation of COL4A4 was confirmed by mutation analysis, and the mutation of c.1826delC was verified by Sanger sequencing. No mutations on COL4A3 and COL4A5 were detected in this family. And the mother and brother are normal wide-type. Conclusions This novel mutation is a valuable addition to the current genetic profile of Alport syndrome, and provide us a better understanding of the disease. Whole-exome sequencing is a power tool to identify the novel mutations of inherited disease and contribute to the molecular diagnosis of disease.

Objective To investigate the effects of hepatocyte growth factor (HGF) on proliferation and transdifferentiation of human Tenon capsule fibroblasts induced by transforming growth factor-β1(TGF-β1) in vitro.Methods Human Tenon capsule fibroblasts were cultured and divided into blank control group,TGF-β1 treated group and different concentrations HGF+TGF-β1 groups.The TGF-β1 at 10 μg/L was added into culture medium of the TGF-β1 treated group,and different concentrations of HGF (25,50,100,200 μg/L) and 10 μg/L TGF-β1 were added into culture medium of the HGF25 μg/L +TGF-β1,HGF50 μg/L +TGF-β1,HGF100 μg/L +TGF-β1,HGF200 μg/L +TGF-β1 group respectively,Methyl thiazolyl tetrazolium (MTT) was employed to measure the cell proliferation (absorbance at 560 nm).Immunofluorescence staining was used to evaluate and locate the expression of α-smooth muscle action (α-SMA) in the cells.The expression of α-SMA protein in the cells was detected by Western blot assay.Results Cultured cells showed fusiform in shape with the positive response for vimentin.The proliferation value of the cells was 0.203±0.025,0.497 ± 0.101,0.426 ± 0.062,0.354 ± 0.040,0.272 ± 0.084,0.241 ± 0.011 in the blank control group,TGF-β1 treated group,HGF25 μg/L +TGF-β1 group,HGF50 μg/L +TGF-β1 group,HGF100 μg/L +TGF-β group and HGF200 μg/L + TGF-β1 group,respectively,showing a significant difference among the groups (F =9.21 0,P =0.003).Compared with the TGF-β1 treated group,the proliferation values of the cells were significantly reduced in the blank control group and HGF+TGF-β1 group (all at P＜0.05).Immunofiuorescence staining showed that α-SMA protein mainly located in cytoplasm with the strong red fluorescence in the cells of the TGF-β1 treated group and weak red fluorescence in HGF+TGF-β1 group,and the expression of α-SMA was absent in the blank control group.The percentage of α-SMA-positive cells was (60.0±4.7)％ in the TGF-β1 treated group and (14.3±3.1)％ in the HGF+TGF-β1 group,with significant difference between the two groups (t =19.856,P＜0.001).The relative expression levels of the α-SMA protein in the cells were 0.642 ±0.032,1.330± 0.069 and 0.884 ±0.040 in the blank control group,TGF-β1 group and HGF100μg/L +TGF-β1 group,respectively,showing a significant difference among the groups (F =13.370,P＜ 0.001),and relative expression levels of the α-SMA protein in the cells were significantly lower in the blank controlgroup and HGF100 μg/L+TGF-β1 group than that in the TGF-β1 treated group (all at P＜0.05).Conclusions HGF can inhibit the proliferation of human Tenon capsule fibroblasts,down-regulate the expression of α-SMA protein induced by TGF-β1 and arrest the phenotype transformation of fibroblasts in vitro.

Objective To investigate the effects of hepatocyte growth factor ( HGF ) on proliferation and transdifferentiation of human Tenon capsule fibroblasts induced by transforming growth factor .β1(TGF.β1) in vitro. Methods Human Tenon capsule fibroblasts were cultured and divided into blank control group, TGF.β1 treated group and different concentrations HGF+TGF.β1 groups. The TGF.β1 at 10 μg/L was added into culture medium of the TGF.β1 treated group,and different concentrations of HGF (25,50,100,200 μg/L) and 10μg/L TGF.β1 were added into culture medium of the HGF25μg/L+TGF.β1 ,HGF50μg/L+TGF.β1 ,HGF100μg/L+TGF.β1 ,HGF200μg/L+TGF.β1 group respectively,Methyl thiazolyl tetrazolium ( MTT) was employed to measure the cell proliferation ( absorbance at 560 nm) . Immunofluorescence staining was used to evaluate and locate the expression of α.smooth muscle action (α.SMA) in the cells. The expression ofα.SMA protein in the cells was detected by Western blot assay. Results Cultured cells showed fusiform in shape with the positive response for vimentin. The proliferation value of the cells was 0. 203±0. 025,0. 497±0. 101,0. 426±0. 062,0. 354±0. 040,0. 272±0. 084,0. 241±0. 011 in the blank control group, TGF.β1 treated group,HGF25μg/L+TGF.β1 group,HGF50μg/L+TGF.β1 group,HGF100μg/L+TGF.β1 group and HGF200μg/L+TGF.β1 group,respectively,showing a significant difference among the groups (F=9. 210,P=0. 003). Compared with the TGF.β1 treated group,the proliferation values of the cells were significantly reduced in the blank control group and HGF+TGF.β1 group ( all at P<0. 05 ) . Immunofluorescence staining showed that α.SMA protein mainly located in cytoplasm with the strong red fluorescence in the cells of the TGF.β1 treated group and weak red fluorescence in HGF+TGF.β1 group,and the expression of α.SMA was absent in the blank control group. The percentage of α.SMA.positive cells was ( 60. 0 ± 4. 7 )％ in the TGF.β1 treated group and ( 14. 3 ± 3. 1 )％ in the HGF+TGF.β1 group, with significant difference between the two groups (t=19. 856,P<0. 001). The relative expression levels of the α.SMA protein in the cells were 0. 642±0. 032,1. 330±0. 069 and 0. 884±0. 040 in the blank control group,TGF.β1 group and HGF100μg/L+TGF.β1 group, respectively, showing a significant difference among the groups ( F=13. 370, P<0. 001),and relative expression levels of the α.SMA protein in the cells were significantly lower in the blank control group and HGF100μg/L+TGF.β1 group than that in the TGF.β1 treated group (all at P<0. 05). Conclusions HGF can inhibit the proliferation of human Tenon capsule fibroblasts, down.regulate the expression of α.SMA protein induced by TGF.β1 and arrest the phenotype transformation of fibroblasts in vitro.

Objective To investigate the related risk factors of the previous cemented vertebral body re-wedge after percutaneous kyphoplasty (PKP).Methods In this retrospective case-control study,clinical data of 617 patients treated by PKP from December 2008 to November 2014 were analyzed.According to whether the cemented vertebra wedged again,the patients were divided into cemented vertebral re-wedge group (n =12) and non-cemented vertebral re-wedge group (n =605).The data of age,preoperative bone density,preoperative vertebral osteonecrosis,intraoperative bone cement injection amount,postoperative bone cement leakage,postoperative bone cement filling,postoperative recovery rate of vertebral wedge angle,and presence or absence of adjacent old vertebral wedging were recorded in two groups.All patients were followed up for one year,and the data were summarized and statistically analyzed.Results Single factor analysis showed the factors of whether there were adjacent old vertebral wedge,preoperative vertebral osteonecrosis,cystic bone cement filling,different wedge angle recovery rate,and emergence of previous cemented vertebral body re-wedge after PKP were statistically significant between two groups (P ＜ 0.05).There was no obvious statistical difference in age,preoperative bone density,intraoperative bone cement injection amount,and intervertebral bone cement leakage between two groups (P ＞ 0.05).Multivariate Logistic stepwise regression analysis showed cystic bone cement filling,preoperative vertebral osteonecrosis,adjacent old vertebral wedging,and higher recovery rate of vertebral wedge angle were prone to appear previous cemented vertebral body rewedge (P ＜ 0.05).Conclusions Relatively higher recovery rate of vertebral wedge angle,previous adjacent old vertebral wedge,vertebral osteonecrosis,and cystic bone cement filling are risk factors closely related to cemented vertebral re-wedge after PKP,which gives a good reference to assess surgical risk,avoid risk factors and choose right surgical techniques.

BACKGROUND:Percutaneous vertebroplasty with bone cement plus hyperextension position reset for acute thoracolumbar vertebral osteoporotic compression fractures has been reported to achieve good clinical efficacy.OBJECTIVE:To evaluate the efficacy of unilateral percutaneous vertebroplasty with bone cement plus hyperextension position reset for Kümmell disease in the elderly.METHODS:Twenty-two old patients with Kümmell disease were included,involving 8 males and 14 females,aged 55-84 years old,and the injuried vertebrae included T11 (n=2),T12 (n=8),L1 (n=7),L2 (n=4),L3 (n=2),L4 (n=2),and L5 (n=1).All patients underwent the unilateral percutaneous vertebroplasty with bone cement plus hyperextension position reset.The visual analogue scale and the Oswestry Disability Index scores,vertebral body height as well as vertebral kyphosis angle were determined before and after treatment.RESULTS AND CONCLUSION:(1) All patients were successfully operated,pain relieved or disappeared within 24 hours postoperatively.No spinal cord injury,pulmonary embolism and other complications were found.One patient presented with cement leakage without obvious clinical symptoms;two patients had non-adjacent vertebral fractures during follow-up.(2) The visual analogue scale and Oswestry Disability Index scores at 1 day postoperatively were significantly lower than those at baseline (P ＜ 0.05).(3) The anterior,medial and posterior height of involved vertebral body,kyphotic angle of involved vertebral body at 1 day postoperatively were significantly higher than those at baseline (P ＜ 0.05).(4) These results manifest that unilateral percutaneous vertebropiasty with bone cement plus hyperextension position reset to treat Kümmell disease in the elderly can significantly relieve back pain,restore partial vertebral height,correct local kyphosis and improve functional recovery of the injured vertebrae.