ObjectiveTo sort out the historical evolution， prescription evolution and modern clinical application of Huagaisan. MethodHuagaisan and its synonym Huagaitang are used as keywords to search the databases of Traditional Chinese Medicine Think Tank， Chinese Medical Dictionary， Airusheng Chinese Medical Database and China National Knowledge Infrastructure（CNKI）. According to the inclusion and exclusion criteria， we obtained the information of ancient books and modern clinical research literature related to Huagaisan， and systematically reviewed and analyzed the historical origin， prescription composition， preparation method， dosage， efficacy， medicinal material origin， processing method and modern clinical application of Huagaisan. ResultA total of 198 pieces of ancient book information were included， involving 93 ancient Chinese medicine books. Huagaisan was composed of fried Perillae Fructus， red Poria， fried Mori Cortex， Citri Eoxcarpium Rubrum， stir-fried Armeniacae Semen Amarum， Ephedrae Herba and fried Glycyrrhizae Radix et Rhizoma， which had the efficacy of promoting the lungs and relieving epidemiological symptoms， expelling phlegm and relieving cough， and treating cough with wind-cold bundled epidemiological symptoms and stagnation of phlegm and Qi. The preparation method was suggested as boiling powder， crushing the seven herbs into coarse particles， the dosage of each drug was fried Perillae Fructus of 1.27 g， red Poria of 1.27 g， fried Mori Cortex of 1.27 g， Citri Eoxcarpium Rubrum of 1.27 g， stir-fried Armeniacae Semen Amarum of 1.27 g， Ephedrae Herba of 1.27 g and fried Glycyrrhizae Radix et Rhizoma of 0.64 g， taking 8.26 g when decocting， adding 300 mL of water， decocting to 210 mL， removing the dregs， and taking it warmly after meals. Twenty-one clinical research papers were included to analyze the modern clinical application of Huagaisan， which was mainly used in the treatment of respiratory diseases such as pneumonia， asthma， bronchitis and so on. ConclusionThis paper has verified and summarized the key information of the famous classical formula Huagaisan， which can provide a detailed reference basis for the development and clinical application of its compound preparation.

Summarize the professor Hu Jingqing's experience in treating pathogenesis of intermingled phlegm and blood stasis by using purgative method.Intermingled phlegm and blood stasis is one of the most important pathogenesis of many difficult diseases in the contemporary era.From the perspective of TCM theory,the purgative method can dissipate phlegm,eliminate blood stasis and resolve masses,which is a suitable method for the treatment in pathogenesis of intermingled phlegm and blood stasis.In clinical practice,the professor Hu Jingqing emphasizes the specific classification of pathogenesis of phlegm and blood stasis in diagnosis,using the purgative methods according to different classifications of"excess heat"and"deficiency cold".And make flexible selections of various purgative drugs in prescription.Last but not least,it is suggested that the purgative method also should not be used as the only treating method.

Pancreatic adenocarcinoma (PAAD) is one of the most lethal malignancies. Although gemcitabine (GEM) is a standard treatment for PAAD, resistance limits its application and therapy. Secoemestrin C (Sec C) is a natural compound from the endophytic fungus Emericella, and its anticancer activity has not been investigated since it was isolated. Our research is the first to indicate that Sec C is a broad-spectrum anticancer agent and could exhibit potently similar anticancer activity both in GEM-resistant and GEM-sensitive PAAD cells. Interestingly, Sec C exerted a rapid growth-inhibiting effect (80% death at 6 h), which might be beneficial for patients who need rapid tumor shrinkage before surgery. Liquid chromatography/mass spectrometry and N-acetyl-l-cysteine (NAC) reverse assays show that Sec C sulfates cysteines to disrupt disulfide-bonds formation in endoplasmic reticulum (ER) proteins to cause protein misfolding, leading to ER stress and disorder of lipid biosynthesis. Microarray data and subsequent assays show that ER stress-mediated ER-associated degradation (ERAD) ubiquitinates and downregulates YAP to enhance ER stress via destruction complex (YAP-Axin-GSK-βTrCP), which also elucidates a unique degrading style for YAP. Potent anticancer activity in GEM-resistant cells and low toxicity make Sec C a promising anti-PAAD candidate.

Objective:To study the effect of continuous subcutaneous insulin infusion (CSII) in the long-term treatment of type 1 diabetes mellitus (T1DM) children, to analyze the factors influencing the efficacy, and to provide theoretical guidance for the application of CSII in long-term therapy and follow-up management.Method:A total of 60 T1DM children who were under 18 years old, lived in Qingdao for a long time, had CSII for more than 6 months, and visited the Outpatient Department of Endocrinology of Qingdao Women and Children′s Hospital for a long term from January 2019 to June 2019 were followed up with questionnaires to understand their general condition and treatment-related data.Result:After the CSII therapy, the hemoglobin A1c (HbA1c) of T1DM children significantly decreased from (9.58±2.08)% to (7.12±1.11)% ( t=7.315, P<0.05), the daily insulin dosage per unit weight significantly declined from 0.91(0.86, 0.94) IU to 0.80 (0.66, 0.88) IU ( Z=-5.514, P<0.05), and the frequency of both hypoglycemia and ketoacidosis was significantly reduced.Diet control, daily exercise time, the visit frequency and the self-monitoring frequency of blood glucose affected the curative effect of CSII therapy (all P<0.05). Conclusion:CSII therapy can effectively control the blood glucose of children with T1DM in Qingdao area, significantly lower HbA1c, and reduce the insulin dosage and the incidence of hypoglycemia and ketoacidosis.Good diet control, regular exercise, regular follow-up and a high frequency of blood glucose monitoring are associated with better glycemic control.

Objective:To investigate the effect of 125I-RSOAds-hTERT/PSA oncolytic adenovirus on targeted therapy of prostate cancer and its effect on tumor microenvironment. Methods:125I-RSOAds-hTERT/PSA ( 125I-virus complex) oncolytic adenovirus was constructed by PCR amplification and double restriction enzyme ligation. TUNEL staining, flow cytometry and Caspase-3 immunoblotting assay were used to detect the killing effect of 125I-RSOAds-hTERT/PSA oncolytic adenovirus on prostate cancer cells in vitro and in vivo, respectively. To explore the effect of 125I-virus complex on tumor tissue cytokine secretion levels, interleukin 2 (IL-2), IL-10, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in the culture supernatant of human prostate cancer cell line PC3, mouse prostate adenocarcinoma cell line RM-1, and mice serum were detected by ELISA. We explored the regulation of 125I-virus complex on the expression of CD24, CD44 and prostate stem cell antigen (PSCA) in prostate tumor tissues and tumor cells through immunohistochemistry. Meanwhile, the expression levels of CD32 and vascular endothelial growth factor (VEGF), as well as CD4+ , CD8+ and macrophage infiltration in tumor tissue were detected through immunofluorescence experiments. Results:125I-virus complex oncolytic adenovirus significantly increased tumor cell apoptosis in vitro and in vivo that was significantly higher than that of 125I group and virus complex group. Meanwhile, IL-2 ( t=-183.30, -38.20, P<0.05), IL-10 ( t=113.80, 92.71, P<0.05), TNF-α ( t=-73.20, -73.91, P<0.05), IFN-γ ( t=-65.37, -139.70, P<0.05) increased in vitro and in vivo. 125I-virus complex reduced the expression of CD24, CD44 and PSCA in tumor cells and tumor tissue, reduced the weight of tumor tissue, inhibited angiogenesis of tumor tissue ( t=8.55, P<0.05), and regulated the immune response in tumor tissue. Conclusions:125I-virus complex targeting prostate cancer can significantly kill cancer cells, reduce the weight and angiogenesis of tumor, and improve tumor microenvironment.

Objective To study the effect and mechanism of Momordica anti-HIV protein of 30 ku (MAP30) on the migration of bladder cancer.Methods The IC50 of human bladder cancer 5637 and T24 cells was calculated by methyl thiazolyl tetrazolium (MTT) method.The migration ability of these two cells was evaluated by scratch migration test and Transwell cell migration test.The expression of migrating proteins such as matrix metalloproteinases (MMPs) and adhesion molecule N-cadherin were compared by Western blot.Results Scratch migration test:there were significant differences in migration rates of 5637 cells at 8 h and 22 h (P ＜ 0.05).There were significant differences in migration rates of T24 cells at 22 h (P ＜ 0.05),but no significant differences in migration rates at 8 h (P ＞ 0.05).The expression of Vimentin,Fibronectin,MMP-2,MMP-9 and N-Cadherin in 5637 cells and T24 cells of human bladder cancer decreased significantly after adding MAP30.The E-Cadherin expression in human bladder cancer 5637 cells were decreased,but no target band was detected in human bladder cancer T24 cells.Conclusions The ribosome-inactivating protein MAP30 can effectively inhibit the migration of human bladder cancer 5637 and T24 cells by inhibiting the EMT pathway and inhibiting the expression of MMPs.

Objective To evaluate the safety and efficacy of telescopic intramedullary rod for treatment of femur fracture or deformity correction in children with osteogenesis imperfecta,and to analysis the result of prevention recurrent fracture as well as the complication.Methods Data of patients who were treated by telescopic intramedullary rod for recurrent femur fracture or curved femoral deformity from March 2015 to December 2015 were prospectively analyzed.There were 39 boys and 26 girls.The average age of the patients was 9 years 2 months,ranging from 3 years 5 months to 13 years 4 month.All the patients had suffered from recurrent femur fractures leading to femoral deformity.The mean angulation angle was 58° (range,30°-95°).Among 69 sides,there were 21 sides of new fracture and 48 sides of deformity.Sixty-one patients were operated at one side and the other 4 patients were treated bilaterally.According to the modified Sillence classification system,there were 5 cases of type Ⅰ,17 type Ⅲ,34 type Ⅴ,3 type Ⅴ,2 type Ⅵ and 4 type ⅩⅤ.Results All the 65 patients were followed up for a mean period of 32 months (range,15-43).The average healing time of the osteotomy site or fracture site of the femur was 8 weeks (range,7-12).The patient was encouraged to begin weight bearing and walking when the Ⅹ-ray film showed healing of the osteotomy or fracture site.By the latest follow up,80％ of the patients could stand and walk independently,The incidence of femur fracture decreased significantly to the level of 0.5±0.2/year,compared to 2.7±1.8/year before operation.All the parents of the children were satisfied with the result of deformity correction.The children's self care and motion ability improved obviously after operation.During follow up,6 patients suffered from recurrent fracture of the femur by various degree,1 of them was treated by open reduction and telescopic rodding surgery,while the other 5 patients were treated conservatively because the fracture displaced or angulated minimally and 4 patients healed uneventfully while 1 patient need plate fixation to augment the axial stability.In 3 patients (1 type Ⅳ,2 type Ⅲ) the intubator failed to elongate with the growth of the distal femoral epiphysis,and in 2 patients the obturator migrated proximally which needed to be re-fixed.Low toxic infections occurred in 2 patients (type Ⅵ) which were treated successfully by removal of the rod and antibiotics.Conclusion The telescopic intramedullary rod can maintain the correction of the femur deformity and improve the quality the bone,thus prevent the recurrent fracture of the femur in children with osteogenesis imperfecta effectively.

Objective To study the effect of enhancer of rudimentary homolog (ERH) gene on migration and invasion in human bladder cancer T24 and 5637 cells.Methods After knocking out the ERH gene of human bladder cancer T24 and 5637 cells,Wound healing assay,Transwell cell migration assay and Transwell cell invasion assay were used to verify the migration and invasion function.Cell migration related protein was detected by Western blot.Nude mouse tail vein transfer assay was used to study the metastasis ability of bladder cancer cells in vivo.Results (1) The Wound healing assay showed that there were significant differences in the migration cell counts of human bladder cancer 5637 and T24 (P ＜ 0.05).(2) There were significant differences in migration and invasion cell counts of Transwell assay (P ＜0.05).(3) Western blot showed that the expression of E-Cadherin in human bladder cancer 5637 cells and T24 cells was significantly increased (P ＜ 0.05) after knocking out ERH gene,while the expression of Fibronectin,Twist,Vimentin and Snail2 protein were significantly decreased (P ＜ 0.05).(4) Nude mouse tail vein transfer assay showed that lung metastases were significantly reduced in the ERH knockout group compared with the normal ERH group.Conclusions Both in vitro and in vivo experiments suggest that ERH knockout affects the migration and invasion of human bladder cancer T24 and 5637 cells.

Objective To investigate the effect of ERH gene knockdown on the proliferation and apoptosis of human bladder cancer T24 cells. Methods T24 cells infected by lentivirus with interference on ERH gene sequence were cloned to establish stable T24 cells clone in ERH gene suppression. The expression of ERH mRNA gene in bladder cancer was detected by using quantitative real time polymerase chain reaction (qPCR). The effects of ERH knockout on the cell proliferation and apoptosis were examined by using methylthiazolyl tetrazolium (MTT) assay, colony formation assay and flow cytometry. The effect of ERH knockout on the tumorigenic effect of T24 cells in vivo was verified by subcutaneous tumor formation in nude mice. Results After lentiviral transfection, qPCR results showed that the knockdown effect of ERH mRNA in ERH normal group (untreated T24 cells) was better than that in ERH gene knockdown group, and the difference was statistically significant [(1.006±0.126) vs. (0.079±0.007); t=12.72, P=0.0002]. After knocking out ERH gene, MTT assay showed that the proliferation ability of T24 cells in ERH gene knockdown group was weakened compared with ERH normal group, and the difference was statistically significant [A490 value: (0.13±0.00) vs. (0.66±0.01);t=104.61, P<0.0001]. Colony formation assay indicated that the ability of clone in ERH normal group was weakened compared with ERH gene knockdown group [(10.5 ±1.2) vs. (196.4 ±4.0); t= 73.63, P< 0.0001]. Flow cytometry showed that the cell apoptosis rate in ERH gene knockdown group was higher than that in ERH normal group [(11.0 ±0.5) ％ vs. (4.2 ±0.5) ％; t= 16.06, P<0.0001]. Imaging results of subcutaneous tumor formation in nude mice showed that the total fluorescence intensity of the tumor area in ERH gene knockdown group was (4.67 ±0.59) × 1010 μW/cm2, and the corresponding part in ERH normal group was (9.54±4.20) × 1010μW/cm2 (t=3.64, P=0.0051);tumor weight in ERH gene knockdown group was (0.80±0.62) g, and in ERH normal group was (1.79±0.71) g (t=3.33, P=0.0037). Conclusion ERH gene knockout can inhibit the proliferation of human bladder cancer T24 cells, and promote the cell apoptosis.

Objective To investigate the carrying status and characteristics of Clostridium difficile isolated from infants.Methods Two hundred and thirty-eight stool specimens were collected from infant younger than 1 year old,that were hospitalized or outpatient from August to November 2015.Immunochromatography targeted GDH and toxin A&B of C.difficile was used for C.difficile screening,and those positive specimens were inoculated in CDIF and anaerobic culture.C.difficile isolates were genotyped by using slpA sequence typing (slpA ST),and tcdA,tcdB,cdtA and cdtB of C.difficile isolates were detected by PCR.Results Fifty C.difficile strains were isolated from 238 stool samples,and the isolated rates of C.difficile from <3 months,3 months to <6 months,and 6 months to 1 years old groups were 9.3%,17.6% and 27.3%(χ2=6.940,P=0.031<0.05),respectively.52.0%(26/50) of the C.difficile isolates were toxigenic,and 69.2% (18/26) toxigenic isolates harbored tcdA+tcdB+cdtA-cdtB-.Fifty C.difficile isolates were genotyped as 11 slpA STs,slpA ST fr-02 and kr-02 were the commonest genotypes in toxigenic C.difficile isolates;however,that was slpA ST xr-03 in non-toxigenic isolates.Conclusion High C.difficile carriage is found in infants younger than 1 year old,and more than half of C.difficile isolates are toxigenic.Most of toxigenic isolates harbored toxin A and B.The genotype of C.difficile isolates is different between toxigenic isolates and non-toxigenic isolates.