Abstract: Objective　To understand the kdr (knockdown resistance, kdr) gene mutation of the voltage-gated sodium channel (VGSC) of Anopheles sinensis in Yunnan Province. Methods　From 2018 to 2019, mosquitoes were collected in Luoping County, Suijiang County, Tengchong City, Yingjiang County, Yuanjiang County and Mengla County in Yunnan Province. The collected mosquitoes were morphologically identified as Anopheles sinensis and genomic DNA was extracted by kits. The DNA templates were sequenced after PCR amplification and the sequencing results were identify as Anopheles sinensis by homology alignment in NCBI. After the ⅡS5 and ⅡS6 fragments of the sodium channels in Anopheles sinensis were amplified and sequenced, the sequencing results were multiple aligned by DNAMAN software, and the mutations were analyzed one by one with BioEdit software to determine the kdr allele types and genotypes, and the frequencies were calculated. Results This survey amplified 287 sequences, and the sequence maps showed that 1014 loci had three alleles, including wild type TTG/L (89.20%), mutant type TTT/F (9.76%) and TCG/S (1.04%). Five genotypes: homozygous wildtype L/L (85.02%), homozygous mutant F/F (6.27%) and S/S (0.35%), heterozygous mutant L/F (6.97%) and L/S (1.39%). The wild type allele TTG/L was the main allele in six sampling sites except Suijiang County. The frequency of wild type allele in Tengchong City was the highest (100.00%). That is, no mutation was detected, while the rest of counties occurred different degrees of mutation at 1014 loci. The frequency of mutant allele in Suijiang County was the highest, reaching 55.68%. Luoping County, Mengla County and Suijiang County had two mutant types. Yingjiang County and Yuanjiang County had one heterozygous mutant L/F. Conclusion　Wild type L1014 (TTG/L) is still dominant in most areas of Yunnan Province. The kdr mutation type is mainly L1014F, followed by L1014S, and the mutation frequency is lower than that in central provinces of China.

OBJECTIVETo observe effects and mechanism of Dinggui gel paste analgesic anti-inflammatory.

METHODSEighty-four male KM mice weighted from 18 to 22 g and aged 4 to 5 weeks were randomly divided into 7 groups, named blank group, model group, matrix control group, Votalin group, high dosage of Dinggui gel paste group with group, equivalent dosage of Dinggui gel paste group, Dinggui gel paste group, 12 mice in each group. Except blank and model group, the other groups were paste ointment for 7 days, and one time a day, matrix control group were pasted isodose blank matrix gel patch. Pain threshold were tested at 30, 60, 90 and 120 min after the last ad-ministration. Hot plate test were performed by injection of 5% formalin for 20 µL on right hindfoot sole after the last administration. The cumulative time of mice licking right rear foot were observed at stage of I and II, and content of IL-1, TNF-α were tested by ELISA method. Differences of weight between right and left ears were measured by ear swelling method and anti-inflammation experiment.

RESULTSIn hot plate test at 90 min, pain threshold in equivalent dosage of Dinggui gel paste group was (24.87 ± 14.67) s and (15.28 ± 8.23) s in model group; (26.33 ± 15.45) s in high dosage of Dinggui gel paste group and (15.31 ± 5.02) s in model group at 120 min in hot plate test, there were no statistical differences between two groups. Pain period at stage I, licking cumulative time in high dosage of Dinggui gel paste group was (66.70 ± 22.83) s and (101.80 ± 33.65) s in model group,and had significant differences between two groups; there were statistical differences in licking cumulative time at stage I of pain period among high dosage of Dinggui gel paste group (51.30 ± 43.60)s, equivalent dosage of Dinggui gel paste group (64.00 ± 47.27) sand model group (109.50 ± 36.78) s. Content of IL-1 in model group was (28.70 ± 8.24) ng/L and (13.33 ± 2.20) ng/L in high dosage of Dinggui gel paste group, there was obvious meaning between two groups; There were significant differences in TNF-α content among model group (93.60 ± 23.65) ng/L,high dosage of Dinggui gel paste group (63.21 ± 10.54)ng/L and equivalent dosage of Dinggui gel paste group (72.69 ± 16.26) ng/L; while there were no statistical meaning in ear swelling degree among model group (5.73 ± 0.80) mg,high dosage of Dinggui gel paste group (5.42 ± 0.68) mg and equivalent dosage of Dinggui gel paste group (4.98 ± 1.52) mg.

CONCLUSIONDinggui gel paste could increase pain threshold, reduce licking accumulative time, and decrease ear swelling degree, and relief pain by regulating level of TNF-α and IL-1.

Analgesics ; administration & dosage ; Animals ; Anti-Inflammatory Agents, Non-Steroidal ; administration & dosage ; Dosage Forms ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Interleukin-1beta ; genetics ; immunology ; Interleukin-6 ; genetics ; immunology ; Male ; Mice ; Ointments ; Pain ; genetics ; immunology ; Pain Management ; Tumor Necrosis Factor-alpha ; genetics ; immunology

Objective To identify the genotypos of extended spectrum β-1actamase (ESBLs)-producing of Escherichia coli ( E. coli) clinical strains isolated from the Macao and compare the results with the genotypes of clinical strains collected in the first Clinical College, China Medical University (CMU) in Shengyang. Methods The clinically isolated E. coli strains including 209 strains from Macao and 150 strains from CMU were collected. Based on the standard of CLSI2006, the ESBLs-producing strains was identified and its isoelectric point(pI) value was detected by isoelectric focusing (IEF) method. The pI values were used to design the primers for PCR amplification. The amplified DNA sequences were then compared with the GenBank and the ESBL genotypes were confirmed. Results ( 1 ) The positive rate of ESBLs-producing strains of E. coli was 30. 1% (62/209) from Macao and 54. 0% (81/150)from CMU. (2)The genotype of 56 (90. 5% ) β-lactamase(ESBLs)-producing E. cull strains from Macao was CTX-M56. Most of them were CTX-M-14 (76. 2% ), other genotypes including CTX-M-9 (4. 8% ), CTX-M-22 (3.2%), CTX-M-24(3.2%), CTX-M-27(1. 6% ), and CTX-M-15( 1.6% ) were found. Six strains were unidentified. (3)The genotype of 74(91.5% )β-lactamase(ESBLs) -producing E. coli strains from Shenyang was CTX-M. Most of them were CTX-M-14 (65.4%), other genotypes including CTX-M-3 ( 13.6% ), CTX-M-24 (4. 9% ),CTX-M-22(2.5%), CTX-M-15(2.5%), CTX-M-9(1.2%) and CTX-M-28(1.2%) were found. Seven strains were unidentified. Conclusions CTX-M genotypo was the mostly identified ESBL-preducing E. Coli strains from Macao and the results were similar with that from CMU. Among them, the CTX-M-14 was the major genotype. Other genotypes included CTX-M-9, CTX-M-15, CTX-M-22, CTX-M-27, and CTX-M-24.However, two genotypes of CTX-M-3 and CTX-M-28 were not found in the clinical isolates in Macao and one genotype of CTX-M-27 was not found from the CMU clinical isolates.

OBJECTIVETo establish a method for real-time recording the oxygen consumption of mice under normobaric hypoxia.

METHODSThe experimental apparatus was made up of animal container, filling water control system, electronic balance, hose, a computer with weight recording software, etc. The working principle was that the oxygen consumed by animal was replaced by water filling which was controlled by the pneumatic and hydraulic actuator. The water was weighted by an electronic balance and the weight signal was recorded into excel file at the same time. The accuracy and precision of the apparatus were detected by a 10 ml syringe. The oxygen consumption characteristics of 6 acute repetitive hypoxia mice and 6 normal mice were observed.

RESULTSThe P value for the paired t test was 1 and the CV value was 4%. The survival time and total oxygen consumption of acute repetitive hypoxia mice were both significantly increased compared to normal mice (P < 0.05), which were (58.8 +/- 6.8) min and (46.0 +/- 8.7) min respectively for the survival time and (85.1 +/- 8.5) ml and (73.6 +/- 5.4) ml respectively for total oxygen consumption.

CONCLUSIONThe hypoxia tolerance of the acute repetitive hypoxia mice can significantly improved by taking more oxygen in the animal cabin. The accuracy and precision of the apparatus are high and it can be used for the determination of oxygen consumption in hypoxia research.

Animals ; Hypoxia ; physiopathology ; Mice ; Monitoring, Physiologic ; instrumentation ; Oxygen Consumption ; physiology

OBJECTIVETo observe mid- and long-term changes in the histopathology and electron microscopic characteristics of the acellular dermal matrix engrafted with thin split-thickness skin autograft.

METHODSTwenty-three biopsy samples were collected from 17 patients undergoing extremity scar resection, who received subsequent grafting using allogenic dermal matrix dressed with thin split-thickness skin autografts. Six months to 2 years after the grafting, the grafts were sampled for histopathological and electron microscopic observations of the layer of the epidermis, thickness of the basal membrane, structural components of the dermis, and infiltration of fibroblasts and revascularization. The data were compared with those of the normal skin samples from the patients.

RESULTSOnly the number of epidermal layers showed statistically significant difference between the skin grafts and the normal skin (16.33-/+5.89 vs 26.57-/+3.46, P=0.007). The thickness of the basal membrane of the skin grafts was similar to that of normal skin, and no significant difference was found in the number of fibroblasts and newly generated capillaries between them.

CONCLUSIONThe mid- and long-term histopathology and ultrastructures of the composite skin graft in the extremities are similar to those of normal skin, suggesting satisfactory effect of the skin grafts.

Adolescent ; Burns ; surgery ; Child ; Child, Preschool ; Cicatrix ; surgery ; Dermis ; transplantation ; ultrastructure ; Female ; Follow-Up Studies ; Graft Survival ; Humans ; Male ; Skin ; ultrastructure ; Skin Transplantation ; methods ; Skin, Artificial ; Transplantation, Autologous

0.05).Moreover,the growth curves of the two kinds of cells were similar.Con- clusions The cell growth properties of cultured transplanted rabbit SMG are similar to that of normal SMG,the cytobiological charac- teristic of transplanted autologous free rabbit SMG are not changed evidently.

OBJECTIVE To investigate the effect of LW- AFC, a new formula of the main active components extracted from Liuwei Dihuang decoction, on treatment of Alzheimer disease (AD) in mouse models. METHODS After treatment LW- AFC, mice were cognitively evaluated in behavioral experiments. Neuron loss, amyloid-β(Αβ) deposition, and Αβ level were analyzed using Nissl staining, immunofluorescence, and an AlphaLISA assay, respectively. Multiplex bead analysis, a radioimmunoassay, immunochemiluminometry, and an ELISA were used to measure cytokine and hormone levels. Lymphocyte subsets were detected using flow cytometry. RESULTS LW-AFC ameliorated the cognitive impairment observed in APP/PS1 mice, including the impairment of object recognition memory, spatial learning and memory, and active and passive avoidance. In addition, LW-AFC alleviated the neuron loss in the hippocampus, suppressed Αβ deposition in the brain, and reduced the concentration of Aβ1- 42 in the hippocampus and plasma of APP/PS1 mice. LW-AFC treatment also significantly decreased the secretion of corticotropin-releasing hormone and gonadotropin-releasing hormone in the hypothalamus, and adrenocorticotropic hormone, luteinizing hormone, and follicle- stimulating hormone in the pituitary. Moreover, LW-AFC increased CD8+CD28+T cells, and reduced CD4+CD25+Foxp3+T cells in the spleen lymphocytes, down- regulated interleukin(IL)- 1β, IL- 2, IL- 6, IL- 23, granulocyte- macrophage colony stimulating factor, and tumor necrosis factor-α and -β, and up-regulated IL-4 and granulocyte colony stimulating factor in the plasma of APP/PS1 mice. CONCLUSION LW-AFC ameliorated the behavioral and pathological deterioration of APP/PS1 transgenic micevia the restoration of the NIM network to a greater extent than either memantineor donepezil, which supports the use of LW-AFC as a potential agent for AD therapy.

OBJECTIVE To observe the anti-aging effects of SOD mimicAEOL-10150 in anti-senescence accelerated mouse resistant 1 (SAMR1) strain. METHODS The lifespan of SAMR1 mice were observed by subcutaneous injection AEOL-101502 mg·kg- 1 once a week. Morris water maze, new object recognition, nesting and forced swimming were used to observe the behavioral changes of animals. Lymphocyte subgroups and ROS were measured by Flow cytometry. The cytokines levels were determined by Luminex method. The number of DCX + neurons in brain tissue was observed by immunofluorescence. RESULTS The results showed that AEOL-10150 could prolong the mean lifespan of SAMR1 mice, but it had no obvious effect on maximal lifespan. What's more, AEOL-10150 could significantly improve the spatial learning memory of aged mice, but it could not increase the number of DCX+ neurons in the hypothalamic MBH and hippocampal DG regions. Then, we observed the effects of AEOL-10150 on peripheral blood lymphocyte subgroups and cytokines. We found that AEOL-10150 significantly modulated the lymphocyte subgroups and cytokine release. Especially, AEOL-10150 can dose-dependently inhibit plasma levels of SASP related inflammatory cytokines TNF-α and IL-17. CONCLUSION The results indicate that AEOL-10150 has anti-aging effects, and the effects are closely related to modulating immunity and inhibiting SASP production.

OBJECTIVE Alzheimer disease(AD),the most common cause of dementia among older people, could not be prevented, halted, or reversed up till now. A large body of pharmacological study has revealed that Liuwei Dihuang (LW) possesses potential therapeutic effects on AD. LW-AFC is key fractions from LW.In the present study,we investigated the effect of LW-AFC on AD mouse models. METHODS PrP-hAβPPswe/PS1ΔE9(APP/PS1) mice and senescence-accelerated mouse prone 8 strain (SAMP8), classic AD animal models, were employed. After the treatment of LW-AFC, mice were cognitively evaluated in behavioral experiments. Neuron loss, amyloid-β (Αβ) deposition, and Αβ level were analyzed using Nissl staining, immunofluorescence, and an AlphaLISA assay, respectively. Multiplex bead analysis, a radioimmunoassay, immunochemiluminometry, and an ELISA were used to measure cytokine and hormone levels.Lymphocyte subsets were detected using fl ow cytometry. RESULTS LW-AFC ameliorated the cognitive impairment observed in APP/PS1and SAMP8 mice,including the impairment of object recognition memory,spatial learning and memory,and active and passive avoidance. In addition, LW-AFC alleviated the neuron loss in the hippocampus, suppressed amyloid-β(Αβ)deposition in the brain,and reduced the concentration of Aβ1-42in the hippo-campus and plasma of APP/PS1 mice. LW-AFC treatment also significantly restored the imbalance of hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axis, enhanced the proliferation of splenocytes,corrected the disorder of lymphocyte subsets,and regulated the abnormal production of cytokine in APP/PS1 and SAMP8 mice. Effects of LW-AFC on pharmacodynamics and neuroendocrine immunomodulation network in APP/PS1 and SAMP8 mice were better than meman-tine and donepezil. CONCLUSION LW-AFC ameliorated the behavioral and pathological deterioration of AD mouse models via the restoration of the NIM network, which supports the use of LW-AFC as a potential agent for AD therapy.

Objective To observe protective effects on rat serum cardiac enzymes and the antioxidant capacity of selenium and vitamin E.Methods According to body weight and 2 × 2 factorial design,eighty male Wistas rats were randomly divided into four groups:low selenium and low vitamin E group(feed containing 23.42％ of the low selenium yeast,excluding vitamin E),low selenium and adequate vitamin E group (feed containing 23.42％ of the low selenium yeast and vitamin E 160 mg/kg),adequate selenium and low vitamin E group(feed containing 46.84％ of the low selenium yeast and sodium seleni 0.25 mg/L in water,excluding vitamin E),adequate selenium and adequate vitamin E group(feed containing 46.84％ of the low selenium yeast,vitamin E 160 mg/kg and sodium selenite 0.25 mg/L in water),20 rats every group.Rats were feed with synthetic feed,and given intraperitoneal anesthesia after 26 weeks of feeding.Blood was collected to observe the impact of selenium and vitamin E on rat cardiac enzymes and myocardial antioxidant capacity and their interactions.Serum creatine kinase (CK) was measured using the continuous monitoring method,creatine kinase isozymes (CK-MB) and lactate dehydrogenase(LDH ) using the immune suppression method,the whole blood GSH-Px assay using the dithiobis nitrohenzoic acid(DTNB) method,serum superoxide dismutase(SOD) using the xanthine oxidase method,total antioxidant capacity (T-AOC) using the complex colorimetry method,the content of propylene glycol (MDA) using the thiobarbituric acid colorimetric method,and reactive oxygen species(ROS) using the colorimetric method.Results Group differences of serum CK,CK-MB,LDH,whole blood GSH-Px activity,serum T-AOC vitality,MDA and ROS content were statistically significant(F=9.797,17.041,48.399,3.744,224.900,49.384,5.045,all P＜ 0.05).Compared with the two low selenium groups and one adequate selenium group,the vitalities of CK,CK-MB,LDH and the contents of MDA[(1577.75 ± 451.87),(1239.15 ± 344.99),(884.25 ± 133.84)U/L,(5.688 ±1.169) × 103 nmol/L; (1474.21 ± 398.38),(1014.84 ± 215.40),(523.00 ± 98.05)U/L,(4.035 ± 0.487 ) × 103 nmol/L and (1180.10 ± 245.51),(948.75 ± 173.68),(676.70 ± 193.63)U/L,(3.406 ± 0.146) × 103 nmol/L]increased significantly in adequate selenium and adequate vitamin E group[( 1056.80 ± 250.98),(721.70 ±129.98),(404.65 ± 72.49)U/L,(3.010 ± 1.270) × 103 nmol/L,all P ＜ 0.05) ].The activity of GSH-Px was obviously increased in the two adequate selenium groups[ (96.611 ± 8.238) × 103,(103.024 ± 8.217) × 103 U/L,all P ＜ 0.05],compared with the two low selenium groups[ (60.356 ± 8.179) × 103,(63.117 ± 8.281) × 103 U/L].Selenium affected the activities of CK,CK-MB and LDH(F =27.09,31.58,29.66,all P＜ 0.01 ),and vitamin E affected the activities of CK-MB and LDH(F=18.9,11.2.all P＜ 0.01 ),but both selenium and vitamin E had no interactions on the activities of CK,CK-MB and LDH (F=0.02,0.001,2.22,all P＞0.05).Selenium affected the activity of GSH-Px and the content of MDA(F=6.74,95.68,all P＜ 0.05),vitamin E affected the activity of T-AOC,the contents of MDA and ROS(F=6.42,36.73,8.43,all P＜0.05),but selenium and vitamin E had interactions only on the content of MDA(F =13.82,P＜ 0.05).Conclusions Long-term selenium or vitamin E deficiency,can reduce the body's antioxidant capacity,leading to the occurrence of myocardial injury.Selenium and vitamin E can improve the body's oxidation capacity,playing a role in myocardial protection.