Objective:To preliminarily explore the role of silent information regulator 3 (SIRT3) in ferroptosis induced by high glucose in renal tubular epithelial cells, and to provide a new theoretical basis and treatment ideas for renal tubular injury in diabetic kidney disease patients.Methods:The single-cell transcriptomic analysis from "Tabula-muris" database was used to evaluate the expression of SIRT3 gene in different cellular subtypes of kidney tissues. HK-2 cells, a human immortalized proximal tubule epithelial cell line, were cultured in vitro and divided into following groups: (1) control group, mannitol group and high glucose group; (2) control group, negative control group, SIRT3 overexpression group, high glucose group and SIRT3 overexpression + high glucose group; (3) control group, negative control group, SIRT3 knockdown group, high glucose group and SIRT3 knockdown + high glucose group; (4) control group, Erastin intervention group and SIRT3 overexpression + Erastin intervention group. Normal glucose was 5.5 mmol/L, high glucose was 30 mmol/L, mannitol was 24.5 mmol/L, Erastin was 10 μmol/L, and the intervention time was 48 h. Cell counting kit-8 proliferation and cytotoxicity assay was used to determine cell viability. Real-time quantitative PCR and Western blotting were performed to assess the expression of SIRT3, kidney injury molecule-1 (KIM-1), and ferroptosis-related proteins acyl-CoA synthetase long chain family member 4 (ACSL4) and glutathione peroxidase 4 (GPX4) at the mRNA and protein levels. The malondialdehyde, glutathione, and iron levels were measured to evaluate the degree of cellular ferroptosis. DCFH-DA was used to analyze the intracellular reactive oxygen species level, while the JC-1 staining method was employed to evaluate alterations of mitochondrial membrane potential in HK-2 cells. Results:(1) The results of single-cell transcriptomic database analysis demonstrated that SIRT3 gene was expressed at the highest level in the subtypes of proximal tubule epithelial cells of kidney tissues. (2) Compared with the control group, the expression levels of KIM-1 and ACSL4 were higher, and the expression levels of SIRT3 and GPX4 and cell viability were lower in the high glucose group (all P<0.05), while there was no statistically significant difference of the aforementioned indicators between the mannitol group and the control group (all P>0.05). (3) Compared with the high glucose group, HK-2 cell vitality, GPX4 expression and intracellular glutathione were higher, ACSL4 expression, intracellular iron, malondialdehyde and reactive oxygen species were lower, mitochondrial membrane potential partially recovered in SIRT3 overexpression + high glucose group (all P<0.05). Compared with the high glucose group, HK-2 cell vitality and GPX4 expression were lower, ACSL4 expression was higher in SIRT3 knockdown + high glucose group (all P<0.05), and there were no statistically significant differences in intracellular iron, malondialdehyde and glutathione (all P>0.05). (4) Compared with the control group, Erastin intervention group had upregulated ACSL4 expression and downregulated GPX4 expression in HK-2 cells (all P<0.05). Compared with the Erastin intervention group, SIRT3 overexpression + Erastin intervention group had upregulated GPX4 expression and downregulated ACSL4 expression (all P<0.05). Conclusions:High glucose can decrease SIRT3 expression and mitochondrial membrane potential, and increase oxidative stress and ferroptosis in HK-2 cells. Overexpression of SIRT3 may reduce oxidative stress and alleviate mitochondrial dysfunction, thereby mitigating glucose-induced ferroptosis in HK-2 cells.

Abstract OBJECTIVE To establish an efficient and simple HPLC-MS/MS method for determination of flucloxacillin in newborn plasma, and to investigate the interaction between ambroxol and flucloxacillin in newborns. METHODS The samples were analyzed by API4000 HPLC-MS/MS. Ultimate XB-C18 column(2.1 mm×100 mm, 5 μm) were carried out. The mobile phase was composed of water-0.1% formic acid(A) and acetonitrile-0.1% formic acid(B). The quantitative analysis of the ion transitions were monitored at m/z 452.6→284.2 for flucloxacillin and m/z 821.4→397.3 for rifampicin(internal standard). RESULTS The linear range of flucloxacillin under this analysis method was 0.20-80 ng·mL-1, and the lower limit of quantification was 0.20 ng·mL-1; The intra-day and inter-day precision of flucloxacillin were both less than 8.23%; The extraction recovery was in the range of 85.3%-89.2%, and the matrix effect was between 89.3%-92.3%; The stability of plasma samples was good under conditions of 12 h at room temperature, 4 h at room temperature after treatment, repeated freeze-thaw for 3 times, and -20 ℃ freezing for 30 d. The results of clinical samples indicated that the combination of ambroxol could significantly increase the blood concentration of flucloxacillin. CONCLUSION The established HPLC-MS/MS method is accurate, sensitive and can be used for the determination of flucloxacillin concentration in neonatal plasma. The results of clinical samples indicate that ambroxol can significantly increase the blood concentration of flucloxacillin. There are drug interactions between ambroxol and flucloxacillin.

OBJECTIVE：To study the effects o f CYP2C9*3 gene polymorphism on therapeutic efficacy of benzbromarone in lowering uric acid and its hepatotoxicity. METHODS ：A retrospective study was conducted to analyze the relevant clinical indicators and genotypes of 196 gout patients who received benzbromarone and CYP2C9*3 gene polymorphism test in Wuhan third hospital from Jan. 2018 to Sept. 2019. RESULTS ：Among 196 patients，179，15 and 2 patients with CYP2C9*3 genotypes * 1/*1， *1/*3 and * 3/*3 genotypes were found ，respectively，and the distribution of each genotype was in line with Hardy-Weinberg balance（P＞0.05）. Before treatment ，there were no significant differences in the levels of UA ，Scr，ALT，AST and CRP between *1/*1 genotype and * 1/*3+*3/*3 genotype（P＞0.05）. After 4 weeks of treatment ，the UA ，Scr，CRP levels of patients with * 1/*1 genotype as well as the UA and CRP levels of patients with * 1/*3+*3/*3 genotype were significantly reduced ，the UA level of patients with * 1/*1 genotype was significantly lower than that of patients with * 1/*3+*3/*3 genotype（P＜0.05 or P＜0.01）. The ALT and AST levels had no obvious changes in patients with different genotype before and after treatment ，and they were in the normal range. No serious abnormal liver function was observed during the treatment. CONCLUSIONS ：Therapeutic efficacy of benzbromarone in lowering uric acid in gout patients with CYP2C9*3 genotypes * 1/*1 genotype is better than that of * 1/*3 and * 3/*3 genotypes. However ，the gene polymorphism may be not associated with its hepatotoxicity.

Objective To study patient delay and the influencing factors of the elderly patients with pulmonary tuberculosis（PTB），so as to provide evidence for developing effective prevention and control strategies. Methods Derived the information from PTB management information system in 2010-2019 of the tuberculosis patients who were aged 60 years or older in Huai'an City, described and analyzed the influencing factors of patient delay. Results The median time of PTB patients delay in Huaian was 21 day, while the rate was 65.91%. Multivariate logistic regression analysis results showed that compared with permanent residents, city dwellers, the first diagnosis unit specialized hospital, and the patient source referral, the patient delay risks of the floating residents（OR = 2.942 , 95% CI: 2.461-3.518）, the country dwellers（OR = 1.528,95% CI :1.377-1.697）, the first diagnosis unit general hospital（OR = 1.203,95% CI: 1.087-1.333）, and the patient source recommendation （OR = 2.395,95% CI: 1.960-2.928）were higher, Compared with the peasants（OR = 0.315 , 95% CI : 0.213-0.512）, new patients（OR = 0.812 , 95% CI : 0.689-0.974） and sputum smear positive patients（OR = 0.866 , 95% CI : 0.780-0.962）, the patient delay risks of the non-peasants, recurrent patients, and sputum negative patients were lower. Conclusion The patient delay of the elderly patients with pulmonary tuberculosis（PTB）in Huaian was serious, the influencing factors of patient delay were type of household registration, current residence, occupation, type of first-time unit, source of patient , classification of treatment, and the sputum test results.

Objective: To evaluate the role of hippocampal CD200 receptor 1 (CD200R1) in perioperative neurocognitive disorders (PND) in mice. Methods: Sixty clean-grade male C57BL/6 mice, aged 9-10 months, weighing 32-38 g, were used in the study.The experiment was performed in two parts.Experiment Ⅰ Thirty-six mice were divided into 2 groups (n=18 each) using a random number table method: control group (group C) and PND group.Group C only received isoflurane anesthesia.Partial left lobectomy of the liver was performed under isoflurane anesthesia in group PND.Contextual fear conditioning test was performed at 1, 3 and 7 days after surgery, and the freezing time was recorded.The mice were then sacrificed, and the hippocampus was isolated for determination of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) contents (by enzyme-linked immunosorbent assay) and CD200 and CD200R1 expression (by Western blot). Experiment Ⅱ Twenty-four mice were divided into 2 groups (n=12 each) using a random number table method: CD200-Fc group and IgG1-Fc group.Recombinant proteins CD200-Fc and human IgG1-Fc were injected into the lateral cerebral ventricle in CD200-Fc group and IgG1-Fc group, respectively.Partial left lobectomy of the liver was performed after the end of injection.Contextual fear conditioning test was performed at 1 and 3 days after surgery, and the freezing time was recorded. Results: Experiment Ⅰ Compared with group C, the freezing time in the contextual fear conditioning test was significantly shortened, and the contents of IL-1β were increased at 1 and 3 days after surgery, the contents of TNF-α were increased at 3 and 7 days after surgery, and the expression of CD200 and CD200R1 was up-regulated at 1 day after surgery in group PND (P<0.05). Experiment Ⅱ Compared with IgG1-Fc group, the freezing time in the contextual fear conditioning test was significantly prolonged at 1 day after surgery in CD200-Fc group (P<0.05). Conclusion Up-regulated expression of hippocampal CD200R1 is the endogenous protective mechanism of PND in mice.

Objective To evaluate the role of hippocampal CD200 receptor 1(CD200R1)in peri-operative neurocognitive disorders(PND)in mice.Methods Sixty clean-grade male C57BL/6 mice,aged 9-10 months,weighing 32-38 g,were used in the study.The experiment was performed in two parts.Ex-periment Ⅰ Thirty-six mice were divided into 2 groups(n＝18 each)using a random number table meth-od: control group(group C)and PND group.Group C only received isoflurane anesthesia.Partial left lo-bectomy of the liver was performed under isoflurane anesthesia in group PND.Contextual fear conditioning test was performed at 1,3 and 7 days after surgery,and the freezing time was recorded.The mice were then sacrificed,and the hippocampus was isolated for determination of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)contents(by enzyme-linked immunosorbent assay)and CD200 and CD200R1 expression(by Western blot).ExperimentⅡ Twenty-four mice were divided into 2 groups(n＝12 each)using a random number table method: CD200-Fc group and IgG1-Fc group.Recombinant proteins CD200-Fc and human IgG1-Fc were injected into the lateral cerebral ventricle in CD200-Fc group and IgG1-Fc group,respectively.Partial left lobectomy of the liver was performed after the end of injection.Contextual fear conditioning test was performed at 1 and 3 days after surgery,and the freezing time was recorded.Re-sults Experiment Ⅰ Compared with group C,the freezing time in the contextual fear conditioning test was significantly shortened,and the contents of IL-1β were increased at 1 and 3 days after surgery,the contents of TNF-α were increased at 3 and 7 days after surgery,and the expression of CD200 and CD200R1 was up-regulated at 1 day after surgery in group PND(P＜0.05).ExperimentⅡ Compared with IgG1-Fc group,the freezing time in the contextual fear conditioning test was significantly prolonged at 1 day after surgery in CD200-Fc group(P＜0.05).Conclusion Up-regulated expression of hippocampal CD200R1 is the endogenous protective mechanism of PND in mice.

Objective To compare the effects of Huiru Yizeng granules Huiru yizeng original decoctin on hyperplasia of mammary glands with hyperprolactinemia in serum and pathological morphology of mammary glands tissue.Methods Seventy rats were randomly divided into 7 groups:normal control group,model control group,Huiru yizeng granules group,Huiru yizeng original decoctin group,Rubisanjie group,bromocriptine group and accessories group.After sucessfully modeling hyperplasia of mammary glandsin rats with hyperprolactinemia,Huiru yizeng granules group was administrated 17.22 g·kg-1·d-1,Huiru yizeng original decoctin group was administrated 20.08 g·kg-1·d-1,Rubi sanjie group was administrated 0.416 mg·kg-1·d-1,bromocriptine group was administrated 0.393 mg·kg-1·d-1.These groups were intragastric administration 2 mL every day for 30 consecutive days.The morphology of pathological tissue in mammary gland was observed by microscope.The levels of prolactin(PRL),progesterone(P),estradiol(E2) were determined by ELISA kit.Results Compared with the model group,Huiru yizeng granules group[PRL=(22.74±4.74) pg·mL-1,P=(46.91±2.85) ng·mL-1,E2=(99.96±9.61) pg·mL-1],Huiru yizeng original decoction group[PRL=(28.41±6.37) pg·mL-1,P=(43.91±4.17) ng·mL-1,E2=(105.02±3.05) pg·mL-1] and bromocriptine group[PRL=(23.58±4.10) pg·mL-1,P=(45.99±2.95) ng·mL-1,E2=(98.04±9.98) pg·mL-1]showed significant decrease in PRL,E2 levels,obvious increase in P(P<0.01).In Huiru yizeng granules group,Huiru yizeng original decoction group and bromocriptine group,PRL,P,and E2 returned to normal level after 30 days,and hyperplasia of mammary glands tissue had great ease.Huiru yizeng original decoction group was Ⅰ to Ⅱ hyperplasia,and Huiru yizeng granules group was 0 or Ⅰ hyperplasia.Compared with each other,the effect of Huiru yizeng granules on the mammary gland proliferation inhibition was superior to Huiru yizeng original decoction group.Conclusion On the treatment of hyperplasia of mammary glands and hyperprolactinemia,Huiru yizeng granules were better than Huiru yizeng original decoction.

Objective To study the relationship between intrauterine infection and early neonatal sepsis.Method From October 2015 to September 2016,the clinical data of pregnant mothers and their newborns in Shenzhen Longhua District Central Hospital were collected,and data of Shenzhen People's Hospital from January 2016 to June 2016 were collected.100 pairs of pregnant mothers and their newborns with confirmed or suspected intrauterine infection were selected as the observation group,and another 100 pairs without intrauterine infection during the same period as the control group.The ratio of term infants vs.premature infants was 1∶ 1.The complete blood count (CBC),CD64,procalcitonin (PCT) and C-reactive protein (CRP) were measured in peripheral blood of all mothers on the day of delivery.The CBC,CD64,CRP,PCT,blood culture of both umbilical venous blood and peripheral blood in neonates were examined and the pathological examination of placenta was performed.Result The positive rate of placental pathology and umbilical cord blood culture in observation group were significantly higher than that in the control and the positive rate in preterms was higher than the terms in observation group (P ＜ 0.05).No significant differences existed between term and premature newborns on the positive rate of peripheral blood culture (P ＞ 0.05).The positive rate of blood culture from umbilical cord blood was higher than peripheral blood in observation group (P ＜ 0.05),but no significant difference in control group (P ＞ 0.05).The incidence of septicemia in term and premature newborns in observation group was significantly higher than the control group (P＜ 0.05).The CD64,PCT in mother's peripheral blood and umbilical cord blood,and CRP in mother's blood were all higher than the control group,the differences were statistically significant (P ＜ 0.05),but CRP in umbilical cord blood in both group were similar (P ＞ 0.05).The area under ROC curve of CD64 and PCT in mother's peripheral blood,CD64 and PCT in umbilical cord blood to diagnose early-onset septicemia in newborns was 0.755,0.793,0.852 and 0.811,respectively.Conclusion The risk of neonatal infections is significantly increased because of intrauterine infection.Combination of peripheral and umbilical blood cultures can increase the accuracy of sepsis diagnosis.Both CD64 and PCT in umbilical cord blood and maternal blood can be used as indicators of intrauterine infection with a predictive value in the diagnosis of early-onset neonatal sepsis.

ABSTRACT:Objective To establish a rapid molecular method for the detection of aldehyde dehydrogenase-2 (ALDH2)and investigate the gene polymorphisms of ALDH2?2 and determine whether the polymorphic ALDH2 gene is associated with drinking behavior in a Chinese population.Methods The gene polymorphisms of ALDH2?2 were detected using pyrosequencing,TaqMan Real-time PCR and GeneChip microarray technologies;genotyping of 302 volunteers was performed to assess their genetic associations with alcohol use behavior.Results We developed pyrosequencing,TaqMan Real-time PCR and GeneChip microarray methods to identify ALDH2? 2 polymorphisms.The allele frequency of ALDH2?2 was 20.36% in the Chinese population:16.33% in the alcoholic group and 27.83% in non-drinkers (P=0.001).In contrast,the genotype frequency of heterozygous ALDH2?1/?2 plus homozygous ALDH2?2/?2 was 45.28% in non-drinkers and 32.65% in the alcoholics group (P=0.030). Allele frequency of ALDH2 genotypes differed significantly between our Chinese sample and other ethnic groups in Asia,and it was significantly higher than that in European and American countries.Conclusion The developed pyrosequencing,TaqMan Real-time PCR and GeneChip microarray methods are rapid,accurate,high-throughput, convenient,and reliable for detecting ALDH2 polymorphisms.ALDH2?2 gene can protect against the development of alcoholism.The allele frequency of ALDH2 in this Chinese population differs from that in other ethnic groups.

Objective To optimize the technological conditions of polysaccharide purification from compound Qianyu water decoction. Methods Water extraction and alcohol precipitation, resolution, Sevag method and dialysis method were used to purify polysaccharide.The purity of polysaccharide was measured with the phenol-sulfuric acid spectrophotometry.On the basis of single factor test, effects of redissolved solid-liquid ratio, number of protein removal, and dialytic time on polysaccharide purity of Qianyu were investigated by orthogonal test. Results The best conditions for purification of polysaccharide in Qianyu were as follows: liquid-solid ratio was 1:40(g/mL, W/V), remove protein for 10 times, and dialysis for 18 h.The content of polysaccharide could reach 69.04%, and the transfer rate was 51.84%. Conclusion The optimized purification process was simple and accurate.It can be used for polysaccharide purification in compound Qianyu water decoction.