Study on mechanism of inhibition effect of N 6-methyladenosine methyltransferase-like 3 on vascular calcification in chronic kidney disease through Bax/Bcl-2 / 中华肾脏病杂志

Objective:To investigate the role and mechanism of N 6-methyladenosine (m 6A) methyltransferase-like 3 (METTL3) in vascular calcification (VC) of chronic kidney disease (CKD) through apoptosis-associated protein. Methods:(1) Real-time fluorescence quantitative PCR was used to test METTL3 mRNA in serum of maintenance hemodialysis (MHD) patients. (2) Western blotting was used to detect the expression of METTL3 protein in high-phosphorus stimulated vascular smooth muscle cells (VSMCs), and immunofluorescence double lable was used to observe the distribution of METTL3 and Runt-related transcription factor 2 (Runx2). The METTL3 overexpressed and knockdown plasmids were constructed and transfected into VSMCs. Alizarin red staining was used to detect calcification degree. Western blotting was used to detect the expressions of osteogenic markers [Runx2, bone morphogenetic protein-2(BMP-2), collagen Ⅰ] and apoptosis- related proteins Bax and Bcl-2. (3) SD rats were randomly divided into control group, CKD-VC group and S-adenosylhomocysteine (SAH) intervention group. The calcification of thoracic aorta was evaluated by von Kossa staining, and the protein expressions of Runx2, Bax and Bcl-2 were detected by immunohistochemistry and Western blotting.Results:(1) METTL3 mRNA expression in MHD patients with VC was significantly lower than that in non-VC patients ( P<0.05), and was negatively correlated with coronary artery calcium score ( r=-0.65, P<0.001). (2) The expression of METTL3 in VSMCs stimulated by high phosphorus was decreased and showed a time dependence. Immunofluorescence double label showed that METTL3 and Runx2 were co-expressed in the nucleus. METTL3 was overexpressed in high-phosphorus induced VSMCs, and the expressions of Runx2, collagen I and BMP-2 were significantly decreased, accompanied by the decrease of calcified nodules and Bax/Bcl-2 ratio (all P<0.05). Conversely, METTL3 knockdown aggravated VSMCs calcification by inducing apoptosis. (3) Furthermore, METTL3 inhibitor SAH was administered in vivo, and it was found that inhibition of METTL3 expression significantly increased the calcification of rat thoracic aorta, and the Bax/Bcl-2 ratio and Runx2 expression were up-regulated. Conclusions:Serum METTL3 level is reduced in MHD patients with VC. In vivo and in vitro studies demonstrate that METTL3 inhibits VC in CKD by mediating the apoptosis-related protein Bax/Bcl-2.

Preparation and Study on in vitro Transdermal Characteristics of Tetramethylpyrazine Pellicle / 中国药房

OBJECTIVE：To preliminarily optimize the preparation technology of Ligustrazine pellicle ，and to study its in vitro percutaneous permeation characteristics. METHODS ：With the amounts of PVA- 124，ethyl alcohol ，glycerin，tween-80 and azone as factors ，single factor experiment was used to optimize the Ligustrazine pellicle matrix formulation ；modified scoring standard was used to evaluate the film formation time ，film formation ability ，ductility，uniformity and the presence of bubble. On the basis of the optimal matrix formulation ，the pellicle with different loading amount of ligustrazine （300，250，200，150，100，50 mg/mL） was prepared and its maximum loading amount was investigated. HPLC method was adopted to determine the content of ligustrazine，and methodology investigation was conducted. Isolated back skin of rats were collected ，the percutaneous permeation test was conducted for high ，medium and low loading amount （100，75，50 mg/mL）of Ligustrazine pellicle. At 15，30，45，60， 75，90，120，150，180 min，the sample was taken and the permeation rate of ligustrazine was calculated. RESULTS ：When the amounts of PVA- 124，ethyl alcohol，glycerin，tween-80 and azone were 2.5 g，7.0 mL，1.97 mL，0.07 mL，0.28 mL（in terms of 50 mL formulation amount ），the optimal matrix formulation of Ligustrazine pellicle was obtained. The maximum drug loading amount of ligustrazine was 100 mg/mL. The linear ranges of ligustrazine was 3.125-100 μg/mL. The specificity，precision， reproducibility，recovery and stability investigation of content determination method of ligustrazine were all in line with the requirements（RSD＜2％）. The permeation rate of high ，medium and low loading amount of Ligustrazine pellicle were 608.42， 384.19，158.20 μg（/ cm2·h）. CONCLUSIONS ：According to the optimized formulation ，the prepared Ligustrazine pellicle had a short film forming time ，stable and re liable quality ； the drug-loading amount was up to 100 mg/mL. The pellicle with drug-loading amount of 75 mg/mL had reached the penetration rate range of effective plasma concentration of ligustrazine treatment.

Safety Evaluation of Bee Venom Plastics with Transdermal Administration / 中国药房

OBJECTIVE： To investigate the acute toxicity， long-term toxicity， skin irritation and anaphylaxis of Bee venom （BV） plastics， and to evaluate its preclinical safety. METHODS： The acute toxicity of BV plastics to rats was investigated after administration of high-dose， medium-dose and low-dose （144， 96， 48 mg/kg） of BV plastics. The long-term toxicity of BV plastics was investigated by continuous administration of high-dose， medium-dose and low-dose （72， 48， 24 mg/kg） of BV plastics for 28 days. The irritation of intact and damaged skin in rabbits with 8 mg/kg BV plastics was investigated by using the self-control method of left and right homologous body. The skin anaphylaxis of guinea pig were investigated after sensitized with 15 mg/kg BV plastics on the left back （on 0， 7th， 14th day） and stimulated with 15 mg/kg BV plastics on the right back. RESULTS： During the acute toxicity experiment with BV plastic，the weight of rats and the changes of viscera were normal，and there was no relevant toxic reaction. Long-term toxicity test results showed that no significant pathological changes were observed at 24 h after the last administration； the spleen index  of rats in BV low-dose group， testicular index in middle-dose group and epididymis index in high-dose and middle-dose groups were significantly increased， while PT in plasma of rats in BV medium-dose and low-dose groups was significantly prolonged （P＜0.05）. There were no abnormal changes in organ appearance， other organ index， coagulation index and blood biochemical index. All above indexes became normal at the end of 2-week recovery period. Skin irritation test showed that BV plastics could cause slight erythema and obvious scab on the skin of rabbits which along with little irritation on intact or damaged skin. Skin anaphylaxis test showed that BV plastics produced mild erythema in the skin of guinea pigs， belonging to light allergy. CONCLUSIONS： No acute or long-term toxicity is observed after transdermal administration of BV plastics， which is safe and only causes mild irritation and irritability to skin， indicating there is good safety of the plastic under experiment doses.

Improvement Effects of Bee Venom Plastics on Experimental Cerebral Thrombosis in Rats / 中国药房

OBJECTIVE： To study the improvement effects of Bee venom（BV） plastics on experimental cerebral thrombosis in rats. METHODS： Totally 96 SD rats were randomly divided into sham operation group （normal saline）， model group （plastics blank matrix）， Nimodipine group （positive drug， 4.00 mg/kg） and BV plastics low-dose， medium-dose， high-dose groups （1.67， 3.33， 6.67 mg/kg）， with 16 rats in each group. Rats in sham operation group and Nimodipine group were given medicine intragastrically， while rats in model group and BV plastics groups were given medicine by transdermal smearing. After 5 days of continuous administration， the experimental cerebral thrombosis model was established by ligating the right external carotid artery and pterygomandibular artery， and injecting compound thrombus inducer into the internal carotid artery. The wet mass ratio of right brain to left brain was measured to investigate the degree of brain edema on the infarcted side. The content of Evans blue （EB） in the left and right hemispheres of rats was determined by ultraviolet spectrophotometry to investigate the cerebral vascular permeability. Blood rheology and coagulation function indicators of rats were measured. The pathological changes of brain tissue in rats were observed by HE staining， and the number of survival neuron cells was counted. RESULTS： Compared with the indexes of sham operation group， the cerebral thrombosis model was established successfully. Compared with model group， the area of blue staining in the right brain （infarcted side） of rats in BV plastics groups was significantly reduced， and the right brain/left brain wet mass ratio and the content of EB in the right brain tissue were significantly reduced （P＜0.05 or P＜0.01）. The whole blood viscosity and Casson viscosity of rats in BV plastics groups， and the plasma viscosity of rats in BV plastics medium-dose and high-dose groups decreased significantly （P＜0.01）. PT and APTT of rats were prolonged significantly in BV plastics medium-dose group （P＜0.01）. The pathological changes of brain tissue in rats in BV plastics groups were significantly alleviated. The arrangement of neuron cells was more orderly， the shape and structure of cells were clear， the nucleolus was clear， the membrane was intact， and the number of survival neuron cells was significantly increased （P＜0.01）. CONCLUSIONS： BV plastics can alleviate brain edema， inhibit cerebral vascular permeability， improve hemorheology and coagulation function indicators of rats after the formation of cerebral thrombosis， and alleviate nerve cell injury after ischemia.

Protective effect of Ento-Ⅰplastic against cerebral ischemia-reperfusion injury in rats / 国际药学研究杂志

Objective To research the protective effect of Ento-Ⅰagainst cerebral ischemia-reperfusion injury in rats,and to evaluate its analgesic and anticoagulating effects in mice. Methods The ischemic model was established with line embolism to block the middle cerebral artery of male rats. The 56 rats were randomly assigned into 7 groups of sham-operation,blank-matrix,nor? mal saline,Ento-Ⅰplastic of 3 doses(6.67,3.33,1.67 mg/kg),and ozagrel sodium(8.3 mg/kg,ip). The effect of Ento-Ⅰplastic on anti-cerebral ischemia was measured by nervous function scores and the areas of cerebral infarction were determined by TTC staining for the calculation of cerebral infarction rates. The analgesic effect of Ento-Ⅰplastic was determined with acetic acid-induced twisting experiment. Sixty KM mice were randomly allocated into blank-matrix,aspirin,aspirin-plastic,and Ento-Ⅰplastic of 3 doses(5,10 and 20 mg/kg),the number of mouse twisting were recorded right after intraperitoneal injection of 0.7%acetic acid solution at the time of 1 h after the last administration. Moreover,the anticoagulant activity of Ento-Ⅰplastic was tested by glass capillary method. Re?sults The results of acetic acid-induced twisting experiment displayed that Ento-Ⅰplastic of all 3 dose groups(5,10 and 20 mg/kg) could significantly reduce the number of body torsion and increase the inhibitory rates of twisting,compared with that of blank matrix group(the inhibitory rates of twisting for 3 dose groups were 21.79%,48.89%,and 56.15%,respectively),with dose-response man?ner. According to the results of glass capillary test,the clotting time of mouse blood could be significantly prolonged by mid-(10 mg/kg)and low-dose(5 mg/kg)of Ento-Ⅰplastic with corresponding clotting time of(155.20±54.19)s and(155.80±73.84)s,compared with normal saline group at(92.10 ± 24.61)and blank-matrix group at(80.40 ± 48.09,P<0.05). The experiment results of the isch?emia-reperfusion injury by line embolism method in rats exhibited that Ento-Ⅰplastic in mid-dose(3.33 mg/kg)could significantly re?duce the neurological scores after 24 h of reperfusion injury,from(2.33 ± 0.52)of normal saline group to(1.00 ± 0.00)of mid-dose group(P<0.01). The results from TTC staining revealed that the cerebral infarction rates of normal saline group and blank-matrix group were(24.89±7.24)%and(27.72±7.89)%,respectively,whereas those of 6.67 mg/kg and 3.33 mg/kg group of Ento-Ⅰplastic were(14.01±2.65)%and(14.73±4.94)%,respectively. Compared to the 2 negative-control groups,both the high-and mid-dose of Ento-Ⅰplastic could significantly reduce the cerebral infarction rates after ischemic reperfusion injury in rats (P<0.01). Conclu?sion Ento-Ⅰplastic demonstrates strong analgesic and anticoagulant effects,and could substantially reduce the neurological scores and reduce cerebral infarction rates for ischemia-reperfusion injured rats. These are likely to be the mechanism of action for Ento-Ⅰplastic realizing its anti-cerebral ischemia effect.

The distribution of bacteria strains isolated from urine samples of pregnant women and drug sensitivity analysis / 国际检验医学杂志

Objective To investigate the distribution characteristics and drug resistance of pathogens isolated from pregnant woman′s urine samples.Methods The urine samples of pregnant women who underwent prenatal examination were collected and cultured.VITEK2 automatic bacterial analyzer was used to identify the bacteria strains,and Escherichia coli ,Streptococcus agalac-tiae were tested for their drug susceptibility.Results The positive rates of urine culture were 4.4%和 3.8% respectively in 2011 and 2012.The top 5 most isolated strains were Escherichia coli ,S .agalactiae ,Enterococcus faecalis ,K .pneumoniae and Propeus vulgaris .In 2011 and 2012,the resistance rates of Escherichia coli to ampicillin and piperacillin was more than 50%,ESBLs-produ-cing rate in Escherichia coli had decreased in 2012 than 2011,the resistance rates of S.agalactiae to erythromycin and clindamycin were more than 30%.Conclusion Escherichia coli accounted for the largest proportion in the strains isolated from urine samples of pregnant women,The pathogen of urinary tract infection in pregnant women are still mainly Gram-negative bacteria(Escherichia co-li),the main pathogens common antibiotics are varying degrees of resistance,pathogens all display common drug resistance of var-ying degrees.

Transoesophageal emergency ventricular pacing with esophageal balloon electrode catheter / 老年心脏病学杂志（英文版）

Objective To evaluate the safety and possibility of transesophageal emergency ventricular pacing with esophageal balloonelectrode catheter.Methods Sixty four patients with critical bradyarrhythmias were divided randomly to receive either transesophagealventricular pacing with esophageal balloon electrode catheter(balloon.catheter group,n=32),or transesophageai ventricular pacing withnormal esophageal electrode catheter(normal catheter group,n=32).Pulse width(ms),current output(mA),and the success rate werecompared between the 2 groups.Results Success rate in the balloon catheter group was 87.5%,which was significantly higher thanthat in the nodal catheter group(18.7%,X2=27.7,P＜0.01).The success rate oftransesophageal ventricular pacing with esophagealballoon electrode catheter was 87.5%.The average current output(mA)was significantly lower in the balloon catheter group than thatin the normal catheter group (24.5+2.5 mA VS 53±3.4mA,P＜0.05).Conclusions Esophageal balloon electrode catheter pacing couldbecome an available teclmiqBe for transesophageal emergency ventricular pacing.