Objective To improve the related substances analysis method of cobamamide.And to further research light degradation characteristics of cobamamide.Methods Determined the related substances of solid and aqueous solution of cobamamide after light degradation by high performance liquid chromatography(HPLC) conditions,which is Merck Hibar C18 column (4.6 mm ×250 mm, 5μm), 0.05 mol/L KH2PO4 solution:acetonitrile (90:10) -80% acetonitrile as mobile by gradient elution and detection wavelength 260 nm.And compared their light stability.The main three kinds of light degradation impurities were determinate from LC-MS.Results Gradient elution made the light degradation impurities separate better.The results of precision and reproducibility tests increased to RSD =0.2% (n=6) and RSD =8% (n=5) from RSD =3.8% (n =6) and RSD=38%(n=5). Cobamamide solution was very sensitive to light, the preparation should be strict dark operation.Two of the light degradation impurities were adenosine and hydroxycobalamin, with the relative response factor 2.5 and 0.7.Conclusion New method is specific, durable and reproducible, which can be used for quality control of cobamamide.

Objective To investigate the influencing factors and improve potency methods of chymosin, to verify the stability and applicability of the national standard of chymosin.Methods The effects of different formula milk powder substrate and enzyme concentration on the determination of the activity of chymosin were studied.3 ×3 dose-response parallel line method was established.The results were compared with the different methods of absolute and relative methods.Results The different formula milk powder had a significant effect on the determination of the absolute potency of the activity of chymosin.The concentration of the enzyme was a power function relationship with the milk clotting time.Compared with the absolute potency, reproducibility of the relative potency of the results was better in different laboratories.The suitable doses in 3 ×3 dose-response parallel line method were 0.35,0.44,0.55U/mL.The confidence limit rate was less than 5%.The potency of the national standard of chymosin (140712-201302) was not significantly different between 2013 and 2015.In a certain dose range, the dose-response of the national standard of chimosin and gastropylor complex or lamb'tripe extract was linear, and the two lines were parallel.Conclusion A lot of factors can affect on the potency of chymosin.Relative potency is determinate by reference standard which can eliminate the influence of different substrates, different operators and endpoint judgment on the determination in order to make results have comparability between laboratories.The test design of 3 ×3 dose-response parallel line can control the test deviation better than the single point determination.The stability of the national standard of chymosin(140712-201302) is good, and is suitable for the potency of chymosin of the products of gastropylor complex and the extract of the lamb.

Objective To investigate the effects of high-calorie diet and age on brain function of ApoE-/-mice.Methods A total of 20 adult ApoE mice(8 months old)and elderly ApoE-/-mice(18 months old)were randomly divided into normal diet adult group,normal diet elderly group,high-calorie diet adult group,and high-calorie diet elderly group,with 5 animals in each group.The mice were fed with corresponding standard diet and high-fat diet for 8 weeks.Their body mass was monitored,and blood glucose was detected with glucose tolerance test.The relative contents of NAA and Cho in the hippocampus and hypothalamus were detected by Magnetic resonance spectroscopy.Y-Maze and open field tests were performed to detect cognitive function,and West-ern blotting was applied to detect the expression of synaptic associated protein 25(SNAP-25),synaptophysin,postsynaptic dense protein-95(PSD-95),iNOS and IL-1β.Results Compared with the normal diet adult group,the NAA content in the hippocampus,Cho and NAA contents in the hypothalamus,spontaneous alternation rate,and expression levels of SNAP-25,synaptophysin and PSD-95 in brain tissue(P＜0.05,P＜0.01)were decreased,and the expression of iNOS and IL-1β(P＜0.01)was increased in the high-calorie diet adult group.The normal diet elderly group had reduced contents of NAA in the hippocampus and Cho in the hypothalamus,and expression levels of SNAP-25,synaptophysin and PSD-95(P＜0.05,P＜0.01),and elevated expression of iNOS and IL-1β(P＜0.01)when compared with the normal diet adult group.Compared with the normal diet elderly group,high-calorie diet resulted in decreased Cho and NAA in both hippocampus and hy-pothalamus,central distance/total distance and down-regulation of SNAP-25,synaptophysin and PSD-95(P＜0.05,P＜0.01),and enhanced expression of iNOS and IL-1β(P＜0.01)in the elderly mice.Compared with the high-calorie diet adult group,the high-calorie diet elderly group had reduced NAA in hippocampus,central distance/total distance and average speed,and decreased expression of synaptophysin(P＜0.05,P＜0.01),and increased expression of iNOS and IL-1β(1.61±0.10 vs 1.35±0.13,2.04±0.08 vs 1.54±0.11,P＜0.05,P＜0.01).Conclusion High-calorie diet results in metabolic disorders and neuroinflammation,inhibits the expression of syn-aptic proteins,and thus leads to cognitive dysfunction in ApoE mice.Long-term high-calorie diet and ageing promote the decline of brain function in ApoE mice.

Objective:To investigate the role and mechanism of astrocyte (AS) derived exosomes in protecting brain microvascular endothelial cells (bEnd. 3) from cerebral ischemia reperfusion injury by establishing an oxygen-glucose deprivation/re-oxygenation (OGD/R) model in vitro. Methods:(1) Dual-luciferase reporter gene assay was used to confirm the regulating effect of miR-193b-3p on transient receptor potential cation channel, subfamily M, member 2 (TRPM2). OGD/R model was established by OGD 8 h followed by reoxygenation 24 h in bEnd. 3 cells after being transfected miR-193b-3p mimics/negative sequence (OGD/R+miR-193b-3p mimics group or OGD/R+miR-193b-3p negative sequence group); real-time quantitative PCR (RT-qPCR) was used to detect the miR-193b-3p expression, Western blotting (WB) was used to detect the expressions of TRPM2, cleaved caspase-3, Bax, Bcl-2, ZO-1 and Claudin-5, and flow cytometry was used to detect the apoptosis. (2) AS was extracted from the cerebral cortex of C57BL/6 suckling mice and identified; modeling time was determined by CCK-8 and AS-derived exosomes were extracted by ultracentrifugation from cell supernatant and identified by electron microscopy, particle size analysis, and WB for marker proteins. RT-qPCR was used to detect the miR-193b-3p expression in AS and AS-derived exosomes. Low-expressed miR-193b-3p exosomes were extracted from AS after being transfected miR-193b-3p inhibitory sequence and co-incubated with OGD/R bEnd. 3 cells (group of OGD/R+AS-derived inhibitory sequence exosomes); exosomes were extracted from AS transfecting with miR-193b-3p negative sequence, and co-incubated with OGD/R bEnd.3 cells (group of OGD/R+AS-derived negative sequence exosomes); and normal exosomes were co-incubated with OGD/R bEnd. 3 cells (group of OGD/R+AS-derived exosomes). The miR-193b-3p expression in these 3 groups was detected by RT-qPCR, expressions of TRPM2, cleaved-caspase-3, Bax, Bcl-2, ZO-1, and Claudin-5 were detected by WB, cell apoptosis was detected by flow cytometry, and cell migration ability was detected by scratch test.Results:(1) Dual-luciferase reporter gene assay showed that miR-193b-3p could bind to TRPM2 mRNA. The miR-193b-3p can improve the TRPM2-mediated apoptosis and tight junction reduction in bEnd.3 cells during OGD/R: compared with OGD/R group, OGD/R+miR-193b-3p mimics group had significantly decreased TRPM2, cleaved-caspase-3, and Bax protein expressions, and statistically increased Bcl-2, ZO-1 and Claudin-5 protein expressions ( P<0.05). Flow cytometry further verified the above results: compared with OGD/R group, OGD/R+miR-193b-3p mimics group had significantly decreased cell apoptosis rate (21.34% vs. 13.93%, P<0.05). (2) The extracted exosomes exhibited lipid bilayer cup-like structure with particle size of 126.5 nm (exosome marker proteins: negative Cal and positive CD81 and SG101), indicating successful exosome extraction. After modeling, miR-193b-3p expression in AS and AS-derived exosomes was significantly decreased compared with that in the Control goup ( P<0.05). Compared with the OGD/R group, the group of OGD/R+AS-derived exosomes and group of OGD/R+AS-derived negative sequence exosomes had significantly increased miR-193b-3p expression, statistically decreased TRPM2, cleaved-caspase-3 and Bax protein expressions, and significantly increased ZO-1, Claudin-5 and Bcl-2 expressions ( P<0.05); while compared with those in the OGD/R group, no significant changes in the above protein expressions in group of OGD/R+AS-derived inhibitory sequence exosomes were noted ( P>0.05). Compared with the OGD/R group (18.22%), group of OGD/R+AS-derived exosomes and group of OGD/R+AS-derived negative sequence exosomes had significantly decreased apoptosis rate (14.09% and 13.79%, P<0.05), while group of OGD/R+AS-derived inhibitory sequence exosomes had no significant change (18.41%, P>0.05). Compared with the OGD/R group (13.55%), group of OGD/R+AS-derived exosomes and group of OGD/R+AS-derived negative sequence exosomes had significantly increased migration ability (43.01% and 40.59%, P<0.05), while group of OGD/R+AS-derived inhibitory sequence exosomes had no significant change (16.26%, P>0.05). Conclusion:AS-derived exosomes can suppress TRPM2 protein expression in brain microvascular endothelial cells distantly by miR-193b-3p to improve the brain microvascular endothelial cell injury caused by OGD/R, and then improve OGD/R injury.

Objective:To understand the pathogenic spectrum and clinical characteristics of respiratory syndrome among hospitalized children in Shanghai, to provide reference for disease prevention and clinical diagnosis.Methods:Twenty-two pathogens were detected in 634 lower respiratory tract aspirates by using fluorescence quantitative PCR combined with the determination method of dissolution curve, and chi-square test was used for statistical analysis of different detection rates.Results:Among the 634 inpatient specimens, 555 were positive for pathogen nucleic acid, with a total positive rate of 87.54%. The detected pathogens were mainly Mycoplasma pneumoniae (MP), adenovirus (ADV), respiratory syncytial virus (RSV), influenza A virus (FluA) and Boca virus (BoV), and the ratio of multiple pathogen infection was greater than single pathogen infection, the pathogen type of multiple infection was ADV, RSV, FluA or BoV co-infection with MP; in the age distribution, FluA has a higher detection rate in the " <2 year old" and " 2-5 years old" age groups, and RSV had a significantly higher detection rate in the " <2 year old" age group compared with the other two age groups, the detection rate of coronavirus (CoV) in " 2-5 years old" and " >5 years old" is higher than that in " <2 year old" age group; combined with the analysis of clinical symptoms, RSV was found in non-febrile patients. The detection rate of RSV and PIV in cough patients was higher than that in non-cough patients, and the detection rate of RSV in wheezing patients was higher than that in non-wheezing patients. H3N2 in RSV and FluA had a high probability of causing pneumonia; FLuA, FluB and RSV had obvious epidemic peaks in winter and spring.Conclusions:We should strengthen the continuous monitoring of respiratory pathogens such as MP, ADV, RSV, FluA and BoV for the purpose of better understanding the changes in their epidemiological characteristics and pathogenicity, and provide the scientific data for the prevention and control of respiratory diseases in children.

Objective To compare the clinical safety and efficacy of CT-guided hook-wire localization of≤10mm pulmonary ground-glass nodule(GGN)via different path ways before video-assisted thoracoscopic surgery(VATS).Methods The clinical data of a total of 128 patients with 10 mm pulmonary GGN,who received CT-guided hook wire localization before VATS at The Third Hospital of Mianyang of China between July 2018 and March 2023,were retrospectively analyzed.According to the puncturing localization path way mode,the patients were divided into vertical puncturing group(n=88)and non-vertical puncturing group(n=40).The number of puncturing times,the time spent for puncturing localization,the success rate of puncture,the operation time of VATS,and puncture-related complications of the two groups were recorded.Results No statistically significant differences in the gender,age,smoking history,GGN location,puncture position,nodule size,density characteristics of GGN,emphysema,and nodules-pleura distance existed between the two groups(all P＞0.05).Compared with non-vertical puncturing group,in vertical puncturing group the number of puncturing times was smaller,the time spent for localization was shorter,the incidence of pneumothorax was lower,and the operation time of VATS was shorter,the differences in all the above indexes between the two groups were statistically significant(all P＜0.05);and the subgroup analysis of patients whose GGN was overlapped with rib shadow obtained the same results.Binary logistic regression analysis revealed that non-vertical puncturing and the number of puncturing times were the independent risk factors for the occurrence of pneumothorax.Conclusion CT-guided hook-wire localization of≤10mm pulmonary GGN before VATS is clinically safe and effective.Under the condition when the lesion can be localized within the range of 2.0cm and the shadow overlapping of GGN with the rib and blood vessel can be effectively avoided,vertical puncturing path way mode should be preferred,which can effectively reduce the incidence of pneumothorax and shorten the operation time of VATS.

ObjectiveTo study the effect of Wulingsan on cerebral edema after intracerebral hemorrhage （ICH） in mice and explore the treatment mechanism. MethodsThe mouse model of ICH was established by injection of collagenase into the caudate nucleus. Mice were randomly assigned into the following groups： sham， ICH， intervention before modeling with low-dose and high-dose （3.69， 11.07 g·kg^-1， respectively） Wulingsan， and intervention after modeling with high-dose Wulingsan. The modified neurological severity score （mNSS） was recorded， and the small animal MRI T2 sequential scanning was performed to measure the volume of cerebral hemorrhage after the modeling of ICH in each group. The Y-maze test， open field test， and Morris water maze test were conducted to evaluate the neurological behaviors of mice in each group. Hematoxylin-eosin staining was employed to observe the pathological changes in the brain tissue. Immunohistochemistry was employed to observe the expression of aquaporin 4 （AQP4）， neuronal nuclei （NeuN）， and glial fibrillary acidic protein （GFAP） in the brain tissue. Western blot was employed to determine the protein levels of AQP4， Claudin-5， and zonula occludens-1 （ZO-1） in the hematoma area. ResultsCompared with the sham group， the ICH group showed increases in the mNSS， the cerebral hemorrhage volume， and the escape latency in the Morris water maze test （P<0.01）， decreases in the times of touching the platform and times of entering the quadrant where the platform was located in the Morris water maze test， and reductions in the spontaneous alternation rate in the Y-maze test and the ratio of distance of center travel to total travel distance in the open field test （P<0.01）. Moreover， pathological changes such as cell disarrangement， cell space enlargement， and cell swelling were observed in the ICH group. Immunohistochemistry results showed that the ICH group had higher proportions of AQP4- and GFAP-positive cells and lower proportion of NeuN-positive cells than the sham group （P<0.01）. Compared with the sham group， the ICH group showed an up-regulated protein level of AQP4 and down-regulated protein levels of Claudin-5 and ZO-1 （P<0.01）. Compared with the ICH group， the intervention with Wulingsan decreased the mNSS， the volume of cerebral hemorrhage， and the escape latency in the Morris water maze test （P<0.05， P<0.01）， while increasing the times of touching the platform and times of entering the quadrant where the platform was located in the Morris water maze test， the spontaneous alternation rate in the Y-maze test， and the ratio of distance of center travel to total travel distance in the open field test （P<0.05， P<0.01）. Furthermore， the intervention with Wulingsan alleviated the pathological changes in the brain tissue after ICH， decreased the proportion of AQP4- and GFAP-positive cells （P<0.01）， increased the proportion of NeuN-positive cells （P<0.01）， down-regulated the protein level of AQP4 （P<0.01）， and up-regulated the protein levels of Claudin-5 and ZO-1 （P<0.01）. ConclusionThe intervention with Wulingsan could reduce the neural function score and the cerebral hemorrhage volume， up-regulate the expression of Claudin-5 and ZO-1， and down-regulate the expression of AQP4 to ameliorate the neurological function defect and cerebral edema after ICH， thereby protecting the brain.

ObjectiveTo investigate the mechanism of Zexie Decoction （泽泻汤） in inhibiting neuroinflammation and improving cognitive impairment mediated by high-calorie diet. MethodsTwenty seven C57BL/J mice were randomly divided into control group （n = 9）， model group （n = 9） and Zexie Decoction group （n = 9）. The mice in the model group and the Zexie Decoction group were fed with high-calorie diet to establish the model of cognitive impairment. Meanwhile， the mice in Zexie Decoction group were also fed with 0.36 g/（kg·d）Zexie Decoction， and the mice in the control group and model group were fed with the same volume of normal saline for 8 weeks. The body weight of mice was recorded at the same time every week； after intervention， oral glucose tolerance test （OGTT） and insulin tolerance test（ITT） commenced； the cognitive level of mice was detected by Morris water maze， open field test， new object recognition test and Y maze； magnetic resonance spectroscopy （MRS） was used to detect the expression of N-acetylaspartate （NAA）， choline （CHO）， lactic acid （Lac）， creatine （Cr）， lipid （Lip）， and myoInositol （mI） in left hippocampus， hypothalamus and cortex. Western blotting was used to detect the expression of synaptophysin （SYN）， synaptosome associated protein-25 （SNAP-25）， postsynaptic dense protein-95 （PSD-95）， tumor necrosis factor-α （TNF-α）， nuclear factor kappa B （NF-κB p65） and its phosphorylated form （P-NF-B p65） in mouse brain； Nissl's staining was used to detect the morphological changes of hippocampal neurons. ResultsCompared with the control group， body mass， blood glucose in oral glucose tolerance test， and blood glucose in insulin tolerance test increased in the model group； in the Morris water maze experiment， the total distance travelled and escape latency of the model group mice increased， the time spent in the platform area and the number of times traversing the platform decreased on days 3 and 4； in the open-field experiment， the number of times the model group mice entered the central area， the ratio of the time in the central area to the total time， and the ratio of the distance travelled in the central area to the total distance significantly decreased； in the new object recognition test， the frequency of new object recognition and recognition index were significantly lower in the model group mice； in the Y-maze test， the spontaneous alternation rate of mice in the model group was significantly lower （P＜0.05 or P＜0.01）； in the left hippocampus， hypothalamus， and cortex of mice in the model group， the CHO/Cr， NAA/Cr significantly decreased， and the mI/Cr， Lac/Cr and Lip/Cr significantly increased； SYN/β-actin， SNAP-25/β-actin and PSD-95/β-actin values significantly decreased， and p-NF-κB p65/NF-κB p65 and TNF-α/β-actin values significantly increased in brain tissue （P＜0.05 or P＜0.01）. Compared with the model group， the above indexes of mice in the Zexie Decoction group significantly improved （P＜0.05 or P＜0.01）. The results of Nissl staining showed that compared with the control group， the neurons in the dentate gyrus of the hippocampus in the model group were scattered and sparsely arranged， the density was significantly reduced， the nuclei of the cells had consolidation and shrinkage， the number of Nissl vesicles was reduced， and the staining became lighter； compared with the model group， the density of neurons in the hippocampal dentate gyrus region of the Zexie Decoction group increased， the wrinkling of nuclei improved， the cell gap narrowed， and the arrangement was slightly tight. Concusion The ameliorative effect of Zexie Decoction on cognitive function in mice with high-calorie diet-induced cognitive impairment may relate to the restructuring of glucose metabolism homeostasis， inhibition of neuroinflammation， reduction of neuronal damage， and enhancement of synaptic plasticity.