BACKGROUND: Bistort rhizome is also named as caoheche, which is characterized by clearing heat, relieving convulsion, regulating damp and reducing swelling. Additionally, its water extract is characterized by antiarrhythmia and central inhibition; however, analgesia should be studied further.OBJECTIVE: To observe analgesic effect of polygonum bistorta L. Water extract, and compare with morphine and amidazofen.DESIGN: Completely randomized digital table and randomized controlled animal study.SETTING: Department of Pharmacology, Gannan Medical College.MATERIALS: The experiment was carried out in the Laboratory of Scientific Center of Gannan Medical College from March to May 2004. ① A total of 150 healthy adult Kunming mice were used in the 4 independent experiments. ② Medicines: Polygonum bistorta L. Water extract (Department of Phytochemistry, Shenyang Pharmaceutical University; batch number: 2003061001); morphine hydrochloride solution (Shenyang First Pharmaceutical Factory; batch number: 000305); naloxone hydrochloride solution (Yanqiao pharmaceutical Co. Ltd.; batch number: 20021109).METHODS: ① Effect of polygonum bistorta L. Water extract on twisting-body reaction of mice induced by acetic acid: Forty mice were randomly divided into 4 groups: saline group, 0.10 and 0.15 mg/g polygonum bistorta L. Water extract groups and amidazofen group with 10 in each group. All mice were intraperitoneally injected with 0.02 mL/g saline,0.10 and 0.15 mg/g polygonum bistorta L. Water extract solution and 0.10 mg/g amidazofen, respectively. Fifteen minutes later, mice were intraperitoneally injected with 6 g/L 0.01 mL/g acetic acid glacial to record times of twisting-body reaction within 15 minutes. ② Effect of polygonum bistorta L. Water extract on pain response of mice induced by hot-plate test: Forty female mice were randomly divided into 4 groups:saline group, 0.10 and 0.15 polygonum bistorta L. Water extract groups and morphine group with 10 in each group. All mice were intraperitoneally injected with 0.02 mL/g saline, 0.10 and 0.15 mg/g polygonum bistorta L. Water extract solution and 0.01 mg/g morphine solution, respectively. GJ-8402 hot-plate pain response threshold detector was used in this study; pain response temperature was (55.0±0.5) ℃; pain response after licking hindfoot was regarded as reactive marker; latency of pain response threshold was within 60 s. Pain response was measured at 15, 30 and 60 minutes after administration with hot-plate test. ③ Effect of morphine, naloxone and polygonum bistorta L. Water extract on pain response of mice induced by hot-plate test: Thirty female mice were randomly divided into 3 groups: saline group, naloxone+morphine group and naloxone+polygonum bistorta L. Water extract group with 10 in each group. All mice were intraperitoneally injected with 0.02 mL/g saline, 0.004 mg/g naloxone solution+0.01 mg/g morphine solution and 0.004 mg/g naloxone solution+0.15 mg/g polygonum bistorta L. Water extract solution, respectivelu. Pain response was measured at 15, 30 and 60 minutes after administration with hot-plate test. ④ Effect of polygonum bistorta L. Water extract on pain response of mice induced by electric stimulation: Forty mice were randomly divided into 4 groups with 10 in each group. All mice were intraperitoneally injected with 0.02 mL/g saline, 0.10 and 0.15 mg/g polygonum bistorta L. Water extract and 1 g/L morphine, respectively. Pain response was measured at 20, 35, 50 and 70 minutes after administration with electric stimulus method.MAIN OUTCOME MEASURES: ① Times of twisting-body reaction; ②duration of pain response induced by hot-plate test; ③ analgesic rate induced by electric stimulation.RESULTS: All 150 healthy adult Kunming mice were involved in the final analysis. ① Times of twisting-body reaction: At 15 inutes after administration, times of twisting-body reaction were less in 0.10 and 0.15 mg/g polygonum bistorta L. Water extract group and amidazofen group than those in saline group [(15.1±11.1), (8.0±6.5), (6.3±3.2), (54.1±20.2) times, t=3.532-3.681, P ＜ 0.01]. ② Duration of pain response induced by hot-plate test:At 15, 30 and 60 minutes after administration, durations of pain response induced by hot-plate test were longer in 0.10 and 0.15 mg/g polygonum bistorta L. Water extract group and morphine group than those in saline group (t=2.676-3.683, P ＜ 0.05-0.01). ③ Duration of pain response was longer in naloxone + polygonum bistorta L. Water extract group than that in saline group at each time point after administration (t=2.676-3.563, P＜ 0.05-0.01); however, duration in naloxone + morphine group was close to that in saline group (P ＞ 0.05). ④ Analgesic rate induced by electric stimulation: At 20, 35, 50 and 70 minutes after administration, analgesic rate induced by electric stimulation was higher in 0.10 and 0.15 mg/g polygonum bistorta L. Water extract group and morphine group than that in saline group (t=3.455-3.634, P ＜ 0.01).CONCLUSION: ① Polygonum bistorta L. Water extract has the obviously analgesic effect, whose intensity is close to that of amidazofen and morphine. ② Naloxone, an opiate receptor antagonist, can resist analgesic effect of morphine but not that of polygonum bistorta L. Water extract. This suggests that analgesic effect of polygonum bistorta L. Water extract dose not react through exciting opiate receptor.

Objective To explore the safety and efficacy of policosanol in elder patients with high on-treatment platelet reactivity ( HPR) after drug-eluting stent ( DES) implantation. Methods This study was a prespecified subgroup analysis of the multicenter, randomized SPIRIT trial,in which there were a total of 169 elder patients (≥60 years old) with HPR. Among these patients, 30 patients were in group A ( given clopidogrel 75 mg/d for one year) , 75 patients in group B ( given clopidogrel 150 mg/d for 30 days followed by 75 mg/d until one year ) and 64 patients in group C ( given policosanol 40 mg/d for 6 month and clopidgrel 75 mg/d for one year ) . All patients were treated with aspirin at the same time. The primary endpoint was the reversion rate of HPR at 30 days (reversion was defined as platelet aggregation ﹤65%). The secondary endpoint was 2-year major adverse cardiac events ( MACE ) rate, which included cardiac death, non-fatal myocardial infarction and ischemic symptoms driven target vessel revascularization. The safety endpoint was any bleeding as defined by the Bleeding Academic Research Consortium ( BARC ) definition. Results At 30 days, the reversion rate of HPR in group C was numerically higher as compared with group A ( 42. 9% vs. 23. 3. 0%, P=0. 068 ) , and similar with group B ( 42. 9% vs. 49. 3%, P=0.447). MACE occurred in 4 (13.3%), 5(6.7%) and 3(4.7%) patients in group A, B and C respictively ( P=0. 352). Bleeding events in group A and group C were both markedly lower in comparison to group B (3. 3% vs. 17. 3% vs. 1. 6%, P=0. 001). At the 24-month follow-up, the MACE-free survival rates were not significantly different (95. 3% vs. 93. 3% vs. 86. 7%, P=0. 146). Conclusions For elder patients with HPR, policosanol reduced platelet reactivity to a similar extent in comparison of high maintenance dose of clopidogrel without increasing bleeding risk.

Objective To investigate the incidence rate, onset time and electrophysiological characteristics of rapid eye movement sleep behavior disorder (RBD) and the relationship between RBD and synucleinopathies as well as the electrophysiological diagnostic criteria of RBD in Parkinson' s disease (PD) and multiple system atrophy (MSA). Methods Sleep survey and night video-polysomnography (NPSG)were used to study sleep disturbance of PD and MSA. (1) Subjective sleep assessments: All subjects,including 66 PD patients, 65 age and sex matched healthy controls and 30 MSA patients, completed the sleep questionnaires, and the RBD incidence rate and onset time were got. (2) Objective sleep assessments: 8 PD patients, 13 MSA patients, and 15 age and sex matched healthy controls underwent video-NPSG recording on two consecutive nights. Sleep architect were analyzed. The NPSG characteristics of RBD accompany with PD and MSA were analyzed, and the electrophysiological diagnostic varameters of it were determined. Results Patients with PD or MSA had a higher prevalence of RBD. RBD was found in 59. 1% (39/66) PD patients and 86. 6% (26/30) MSA patients, among those, 46. 2% ( 18/39 ) and 84.6% (22/26) had the waking symptoms of MSA and PD. The main NPSG characteristics of RBD of PD or MSA were chin REM without atonia (RWA) and increased movement. Conclusions The relatively higher RBD prevalence in MSA and PD patients indicates that RBD has close relationship with PD and MSA.Part of patients with RBD preceding neurology disease indicates that RBD may be the early marker of PD and MSA. The main NPSG characteristics of RBD accompany with PD and MSA are chin RWA and the motor manifestations. RWA and phasic EMG activity density are supposed to be the NPSG diagnostic parameters.

Objective To cultivate human leukemia cells (HL-60) which were transiently transfected either a miR-126 mimic or inhibitor,and then to characterize the proliferation and apoptotic behavior of the transfected leukemia cells.Methods The leukemia cell line was developed and RT-PCR was performed to evaluate miR-126 expression levels.An instantaneous plasmid tmnsfection technique was used to transfect cells with either the miR-126 mimic or inhibitor.CCK-8,FCM,and clone formation tests were performed to analyze the proliferative and apoptotic behaviors of the leukemia cells.Results Proliferation was significantly decreased in cells transfected with the miR-126 mimic for 0,24,48,and 72 hours (P ＜ 0.05).Specifically,the G1,S,and G2 phases were significantly inhibited (P ＜ 0.05),the late and early apoptosis (UR+LR) rate increased (P ＜ 0.05),and the average rate of colony formation was also significantly decreased (P ＜ 0.05).Additionally,proliferation was significantly increased in cells transfected with the miR-126 inhibitor for 0,24,48,and 72 hours (P ＜ 0.05).Specifically,the G1,S,and G2 phases were increased (P ＜ 0.05),the UR + LR decreased significantly (P ＜ 0.05),and the average rate of colony formation was significantly increased (P ＜0.05).Conclusion In HL-60 cells,miR-126 can inhibit proliferation and promote apoptosis;thus,miR-126 may play an important role in the occurrence and development of leukemia as a tumor-suppressor miRNA.

Objective:To evaluate the utility of whole-exome sequencing (WES) in early diagnosis for children with language delay/disorder.Methods:Children with language delay/disorder who were admitted to the Department of Health Care, Children′s Hospital Affiliated to the Capital Pediatric Institute from January 2019 to December 2020 were analyzed retrospectively. Based on informed consent, the peripheral blood of the children and their parents was collected for WES. Combining the clinical phenotypes of the children, the candidate variants, including single nucleotide variants (SNVs) and copy number variations (CNVs), were selected for validation and family segregation analysis using Sanger sequencing, real-time PCR or CNV-Seq. The pathogenicity of variants was evaluated based on ACMG guideline following with finial genetic diagnosis. Based on whether genetic diagnosis was achieved or not, 125 children with comprehensive examination of the Children Neuropsychological and Behavioral Scale(CNBS-R2016) were sub-grouped (positive/negative group), and the total scores and the detailed scores of five developmental sections (gross motor, fine motor, adaptive ability, language and social behavior ability) between two subgroups were compared.Results:A total of 165 children with language delay/disorder were recruited, including 109 males and 56 females. The ratio of boys to girls was 1.95∶1.The age of the children was (3.2±1.2) years old, the median age was 3.0 years. 45 children carry disease-related pathogenic/likely pathogenic variants, including 36 SNVs and 9 CNVs. The genetic diagnostic yield of this cohort was 27.3% (45/165). The inheritance analysis for core family members showed de novo variant accounted for 86% of genetic diagnosis (31/36). The positive diagnosis rate in girls was 45% (25/56), which was significantly higher than that in boys (18.3%, 20/109, χ2=12.171, P<0.05). There was no significant difference in the rate of positive diagnosis among all age groups (χ2=4.349, P>0.05). Interestingly, the scores of gross motors of positive group were significantly lower than that of negative group (61.5 vs. 69.4, t=-2.610, P<0.05). Otherwise, no significant difference was seen between two groups( t=-0.933, -1.298, -0.114, -0.214, all P>0.05). Conclusions:Language delay/disorder has complex genetic heterogeneity. WES has important application value in early etiological diagnosis for children with language delay/disorder.

Objective:To evaluate the utility of whole-exome sequencing (WES) in early diagnosis for children with language delay/disorder.Methods:Children with language delay/disorder who were admitted to the Department of Health Care, Children′s Hospital Affiliated to the Capital Pediatric Institute from January 2019 to December 2020 were analyzed retrospectively. Based on informed consent, the peripheral blood of the children and their parents was collected for WES. Combining the clinical phenotypes of the children, the candidate variants, including single nucleotide variants (SNVs) and copy number variations (CNVs), were selected for validation and family segregation analysis using Sanger sequencing, real-time PCR or CNV-Seq. The pathogenicity of variants was evaluated based on ACMG guideline following with finial genetic diagnosis. Based on whether genetic diagnosis was achieved or not, 125 children with comprehensive examination of the Children Neuropsychological and Behavioral Scale(CNBS-R2016) were sub-grouped (positive/negative group), and the total scores and the detailed scores of five developmental sections (gross motor, fine motor, adaptive ability, language and social behavior ability) between two subgroups were compared.Results:A total of 165 children with language delay/disorder were recruited, including 109 males and 56 females. The ratio of boys to girls was 1.95∶1.The age of the children was (3.2±1.2) years old, the median age was 3.0 years. 45 children carry disease-related pathogenic/likely pathogenic variants, including 36 SNVs and 9 CNVs. The genetic diagnostic yield of this cohort was 27.3% (45/165). The inheritance analysis for core family members showed de novo variant accounted for 86% of genetic diagnosis (31/36). The positive diagnosis rate in girls was 45% (25/56), which was significantly higher than that in boys (18.3%, 20/109, χ2=12.171, P<0.05). There was no significant difference in the rate of positive diagnosis among all age groups (χ2=4.349, P>0.05). Interestingly, the scores of gross motors of positive group were significantly lower than that of negative group (61.5 vs. 69.4, t=-2.610, P<0.05). Otherwise, no significant difference was seen between two groups( t=-0.933, -1.298, -0.114, -0.214, all P>0.05). Conclusions:Language delay/disorder has complex genetic heterogeneity. WES has important application value in early etiological diagnosis for children with language delay/disorder.

Objective:To investigate the clinical application and effect of end-to-side anastomosis in personalised free ilioinguinal flap transfer.Methods:From March, 2015 to July, 2020, 88 patients with soft tissue (bone) defect of limbs were treated. Different ilioinguinal flaps were designed according to the wound condition of patients, which were 48 cases of free superficial circumflex iliacartery perforator flap, 7 cases of free superficial epigastric artery perforator flap, 19 cases of composite tissue flap with iliac bone, 8 cases of combined flap of superficial circumflex iliac artery and superficial abdominal wall artery, and 6 cases of superficial circumflex iliac artery and superficial abdominal wall artery lobulated flap. The area of the flap was 4.0 cm×6.0 cm-10.0 cm×30.0 cm. The artery and recipient artery were anastomosed end-to-side: 36 cases to radial artery; 12 cases to ulnar artery; 18 cases to dorsalis pedis artery; 15 cases to anterior tibial artery; 7 cases to posterior tibial artery. Venous anastomosis of skin flap: 42 cases were anastomosed with 2 veins, which were superficial vein of the same name and accompanying vein; 46 cases were anastomosed with 1 superficial vein of the same name. The accompanying vein of the flap was anastomosed end-to-side with the accompanying vein of the main artery of the recipient area, and the superficial vein of the same name was anastomosed end-to-end with the accompanying vein or subcutaneous superficial vein of the recipient artery. Follow-up includes flap blood supply, blood supply to the distal limbs, appearance of both the donor site and the recipient area, and patient satisfaction.Results:There were 83 cases of flaps survived successfully, and 5 cases of crisis. Among them, 2 cases had artery crisis at 48 h after surgery. After exploration, it was found that 1 case caused by arterial thrombosis, and 1 case compressed by the stapler that anastomoses the vein. The other 3 cases had venous crisis at 72 h after surgery: after exploration, it was found that caused by thrombosis at the venous anastomotic site. The average follow-up period was 10 (range, 3-24) months. All flaps survived after re anastomosis or vascular transposition. The donor site and recipient site of the flap healed well. The blood supply of the flap was good and the texture was soft. There was no blood supply disorder in the distal limb.Conclusion:The end-to-side anastomosis technique is suitable for all kinds of free flap transplantation in ilioinguinal region, with high vascular patency rate. It can not only solve the problem of thin vascular pedicle of donor site flap, but also retain the main artery of recipient limb without affecting the distal blood supply.

OBJECTIVETo study the effect of Mps1 on BRAFWT/MEK/ERK pathway in the presence of wild type BRAF or BRAFV600E in melanoma.

METHODSMelanoma cells harboring BRAFWT genotype were transfected either with pBabe-puro-GST-BRAF-WT and/or pBabe-puro-GFP-Mps1-WT or pBabe-puro-GST-BRAFV600E and/or pBabe-puro-GFP-Mps1-WT, followed by Western blot to detect Mps1 and p-ERK expression. The melanoma cells harboring BRAFWT and BRAFV600E genotype were infected with pSUPER-Mps1 retrovirus to knockdown the endogenous Mps1 protein, followed by Western blot to detect Mps1 and p-ERK expression. Meanwhile, melanoma cells harboring BRAFV600E genotype were infected with pBabe-puro-GFP-Mps1 and Western blot was performed to detect Mps1 and p-ERK expression.

RESULTSIn melanoma cells harboring BRAFWT genotype and transfected with pBabe-puro-GST-BRAF-WT and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels were notably reduced as compared to either negative control or empty vector. However, cells transfected with pBabe-puro-GST-BRAFV600E and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels did not change significantly compared with either negative control or empty vector. Knockout of Mps1 in BRAF wild-type cell lines led to an increased ERK activity. However, there was no significant change of ERK activity in BRAFV600E cell lines in the absence of Mps1. The expression of p-ERK in BRAFV600E mutant cell lines infected with pBabe-puro-GFP-Mps1-WT did not show any significant difference from either negative control or empty vector.

CONCLUSIONSBased on these findings, it suggests that there exists an auto-regulatory negative feedback loop between the Mps1 kinase and BRAFWT/ERK signaling. Oncogenic BRAFV600E abrogates the regulatory negative feedback loop of Mps1 on the MAPK pathway.

Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; Humans ; MAP Kinase Signaling System ; Melanoma ; genetics ; metabolism ; Mutation ; Phenotype ; Protein-Serine-Threonine Kinases ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Proto-Oncogene Proteins B-raf ; metabolism ; Signal Transduction ; Transfection

Objective To study the effect of Mps1 on BRAFWT/MEK/ERK pathway in the presence of wild type BRAF or BRAFV600E in melanoma.Methods Melanoma cells harboring BRAFWT genotype were transfected either with pBabe-puro-GST-BRAF-WT and/or pBabe-puro-GFP-Mps1-WT or pBabe-puro-GST-BRAFV600E and/or pBabe-puro-GFP-Mps1-WT, followed by Western blot to detect Mps1 and p-ERK expression.The melanoma cells harboring BRAFWT and BRAFV600E genotype were infected with pSUPER-Mps1 retrovirus to knockdown the endogenous Mps1 protein, followed by Western blot to detect Mps1 and p-ERK expression.Meanwhile, melanoma cells harboring BRAFV600E genotype were infected with pBabe-puro-GFP-Mps1 and Western blot was performed to detect Mps1 and p-ERK expression. Results In melanoma cells harboring BRAFWT genotype and transfected with pBabe-puro-GST-BRAF-WT and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels were notably reduced as compared to either negative control or empty vector.However, cells transfected with pBabe-puro-GST-BRAFV600E and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels did not change significantly compared with either negative control or empty vector.Knockout of Mps1 in BRAF wild-type cell lines led to an increased ERK activity.However, there was no significant change of ERK activity in BRAFV600E cell lines in the absence of Mps1.The expression of p-ERK in BRAFV600E mutant cell lines infected with pBabe-puro-GFP-Mps1-WT did not show any significant difference from either negative control or empty vector. Conclusions Based on these findings, it suggests that there exists an auto-regulatory negative feedback loop between the Mps1 kinase and BRAFWT/ERK signaling.Oncogenic BRAFV600E abrogates the regulatory negative feedback loop of Mps1 on the MAPK pathway.

Objective: To compare the efficacy of Bracka method and Duckett method in the treatment of proximal hypospadias. Methods: Forty patients with hypospadias were treated by 2 stages(Bracka), 42 patients treated by transverse preputial island flap (Duckett) from January 2014 to January 2016. Mean age at first stage surgery were (19.70±6.62) months and (20.33±5.03) months in Bracka group and Duckett group, respectively. There were 10 cases of proximal penile type, 25 cases of penoscrotal type, 5 cases of perineal type in group 1. There were 11 cases of proximal penile type, 27 cases of penoscrotal type, 4 cases of perineal type in group 2. There was no significant difference in age and hypospadias classification between the two groups(P>0.05). All operations were performed by the same doctor. Urethral plate reconstruction with preputial graft was performed in group 1; stage Ⅱ Duplay urethroplasty repair was carried out 6-8 months after stage Ⅰ. Results: Urine tube was placed for 2 weeks after operation and followed up for 36-63 months(mean 47.6 months). After stage I repair, penile straightening and wide, smooth appearance of graft were confirmed. There was no fistula, split, urethral diverticulum or other complications, one case with urethral opening stenosis who was restored after urethral dilatation .After stage II repair, urethral fistula was noted in 3 cases(7.5%), stricture in 1 cases(2.5%). No other complications occurred . The total rate of complications was 10%(10/40). Urethral fistula was noted in 7 cases(16.7%), stricture in 3 cases(7.1%), penile head dehiscence in 3 cases(7.1%) and diverticulum in 1 case(2.4%) in group 2. The total rate of complications was 33.3%(14/42). The incidence of total complications between the two groups was statistically significant (P=0.011). Conclusions Bracka method can be used to treat proximal hypospadias. It has high safety and low incidence of complications.