Objective To investigate the role of Toll-like receptor 4(TLR4)in the regulation of homocys-teine(Hcy)-induced ferroptosis in macrophages.Methods Mouse macrophage cells RAW264.7 were cultured and divided into control group,Hcy intervention group(Hcy group),and Hcy plus ferroptosis inhibitor group(Hcy+Fer-1 group).After transfection with interference fragments,macrophages were treated with Hcy,and then divided into control group,Hcy intervention group(Hcy group),TLR4 interference negative control plus Hcy intervention group(si-NC+Hcy group),and TLR4 interference plus Hcy intervention group(si-TLR4+Hcy group).Macrophages were transfected with overexpression lentivirus and treated with Hcy,then were divided into control group,Hcy intervention group(Hcy group),a TLR4 overexpression negative control plus Hcy intervention group(OE-NC+Hcy group),and a TLR4 overexpression plus Hcy intervention group(OE-TLR4+Hcy group).After 48 hours of intervention,real-time fluorescent quantitative PCR and western blot were used to detect the expression levels of TLR4 in macrophages treated with Hcy;western blot was used to detect the expression levels of ferroptosis-related proteins ACSL4,GPX4,and FTH1 in macrophages,and ferrous ion assay kit to detect the concentration of Fe2+in macrophages;reactive oxygen species(ROS)assay kit and laser confocal microscopy were used to detect the content of intracellular reactive oxygen species.Results Compared with those in the control group,the expression level of the pro-ferroptosis protein ACSL4 was increased in the Hcy group(P<0.05),while the expression levels of anti-ferroptosis proteins GPX4 and FTH1 were decreased(P<0.05);the concentration of Fe2+was increased(P<0.05),and the content of ROS was increased.Meanwhile,the protein and mRNA expres-sion levels of TLR4 were both increased in the Hcy group(P<0.05).After macrophages were transfected with TLR4 interference fragments,compared with those in the si-NC+Hcy group,the expression levels of GPX4 and FTH1 were increased(P<0.05);the expression level of ACSL4 was decreased(P<0.05);the concentration of Fe2+was decreased(P<0.05),and the content of ROS was reduced in the si-TLR4+Hcy group.After macro-phages were transfected with TLR4 overexpression lentivirus,compared with those in the OE-NC+Hcy group,the expression levels of GPX4 and FTH1 were decreased(P<0.05),and the expression level of ACSL4 was increased(P<0.05)in the OE-TLR4+Hcy group.Conclusion Hcy induces the occurrence of ferroptosis in macrophages,and Toll-like receptor 4 has a positive feedback regulatory effect on ferroptosis in macrophages.

Objective To investigate the role of Toll-like receptor 4(TLR4)in the regulation of homocys-teine(Hcy)-induced ferroptosis in macrophages.Methods Mouse macrophage cells RAW264.7 were cultured and divided into control group,Hcy intervention group(Hcy group),and Hcy plus ferroptosis inhibitor group(Hcy+Fer-1 group).After transfection with interference fragments,macrophages were treated with Hcy,and then divided into control group,Hcy intervention group(Hcy group),TLR4 interference negative control plus Hcy intervention group(si-NC+Hcy group),and TLR4 interference plus Hcy intervention group(si-TLR4+Hcy group).Macrophages were transfected with overexpression lentivirus and treated with Hcy,then were divided into control group,Hcy intervention group(Hcy group),a TLR4 overexpression negative control plus Hcy intervention group(OE-NC+Hcy group),and a TLR4 overexpression plus Hcy intervention group(OE-TLR4+Hcy group).After 48 hours of intervention,real-time fluorescent quantitative PCR and western blot were used to detect the expression levels of TLR4 in macrophages treated with Hcy;western blot was used to detect the expression levels of ferroptosis-related proteins ACSL4,GPX4,and FTH1 in macrophages,and ferrous ion assay kit to detect the concentration of Fe2+in macrophages;reactive oxygen species(ROS)assay kit and laser confocal microscopy were used to detect the content of intracellular reactive oxygen species.Results Compared with those in the control group,the expression level of the pro-ferroptosis protein ACSL4 was increased in the Hcy group(P<0.05),while the expression levels of anti-ferroptosis proteins GPX4 and FTH1 were decreased(P<0.05);the concentration of Fe2+was increased(P<0.05),and the content of ROS was increased.Meanwhile,the protein and mRNA expres-sion levels of TLR4 were both increased in the Hcy group(P<0.05).After macrophages were transfected with TLR4 interference fragments,compared with those in the si-NC+Hcy group,the expression levels of GPX4 and FTH1 were increased(P<0.05);the expression level of ACSL4 was decreased(P<0.05);the concentration of Fe2+was decreased(P<0.05),and the content of ROS was reduced in the si-TLR4+Hcy group.After macro-phages were transfected with TLR4 overexpression lentivirus,compared with those in the OE-NC+Hcy group,the expression levels of GPX4 and FTH1 were decreased(P<0.05),and the expression level of ACSL4 was increased(P<0.05)in the OE-TLR4+Hcy group.Conclusion Hcy induces the occurrence of ferroptosis in macrophages,and Toll-like receptor 4 has a positive feedback regulatory effect on ferroptosis in macrophages.

Objective:In this study,three methods of removing microglial cells from primary astrocyte culture were compared and analyzed to explore the characteristics of different purification methods.It provides the basis for research-ers to select the appropriate primary astrocyte culture method.Methods:The cerebral cortex cells of SD neonatal rats were isolated and platted and divided into an unpurified group(control group),a traditional oscillation purification group(TO group),a cytosine β-D-arabinofuranoside(AraC)+TO group,and an AraC+L-leucine methyl ester(LME)group according to different purification methods.Cell counts were used to calculate the cell production after different purification treatments.The cell morphology of each group was observed under an optical microscope,and the proportion of glial fibrillary acidic protein(GFAP)positive cells was calculated by immunofluorescence staining.AlamarBlue HS was used to detect cell viability after passage.Results:Compared with the control group,the cell purity(P＜0.0001)and cell viability(P＜0.0001)of purified cells in the TO group were significantly increased after pas-sage,while the total number of purified cells was significantly decreased(P＜0.001).These trends were more obvious in the AraC+TO and AraC+LME group,and the cell purity(P＜0.0001 or P＜0.0001)and cell viability(P＜0.01 or P＜0.001)of purified cells in the AraC+TO and AraC+LME group after passage were significantly higher than those in the TO group.The total amount of cells obtained was significantly decreased compared with the TO group(P＜0.01 orP＜0.01).There was no significant difference between the AraC+TO and AraC+LME group.Conclusion:The culture rich in astrocytes(cell purity＞90％)was obtained by the TO method,and the cell yield was high,which could be applied to routine cell research.The highly enriched astrocyte cultures(cell purity＞99％)obtained by the AraC+TO and AraC+LME purification methods can be applied to study the precise role of astrocytes in the context of neuroinflammation.

Endovascular treatment has become the standard treatment method for acute ischemic stroke caused by large vessel occlusion. The application of neuroimaging techniques for appropriate patient selection and prognosis prediction is of great significance for successful endovascular treatment. This article reviews the application progress of fluid-attenuated inversion recovery sequence vascular hyperintensity in patients with acute ischemic stroke underwent endovascular treatment.

Objective To explore the role of the cGAS-STING signaling pathway in the therapeutic mechanism of Liangxue Jiedu Huayu Formula(LXJDHYF)for acute-on-chronic liver failure(ACLF)in mice.Methods Thirty C57BL/6 mice were randomly divided into blank control group,model group,low-and high-dose LXJDHYF groups,and H151(a specific cGAS-STING pathway inhibitor)group(n=6).In all but the control group,the mice were treated with CC14 to induce liver cirrhosis followed by intraperitoneal injections of lipopolysaccharide and D-amino galactose to establish mouse models of ACLF.After the treatments,the mouse livers were collected for HE and TUNEL staining,and serum levels of ALT,AST and TBil were determined.In bone marrow-derived macrophages(BMDMs)and liver tissues of ACLF mice,the expressions of cGAS-STING signaling pathway-related mRNAs including IFN-β,ISG15,IL-6 and TNF-α were determined with RT-qPCR,and the phosphorylation levels of IRF3 and STING proteins were investigated using Western blotting.Results Compared with the mice in the model group,the LXJDHYF-treated mice exhibited milder hepatocyte necrosis and inflammatory cell infiltration in the liver with significantly reduced hepatocyte apoptosis.LXJDHYF treatment also significantly lowered serum levels of ALT,AST,TBil,IL-6 and TNF-α in ACLF mice and effectively suppressed the expressions of cGAS-STING signaling pathway-related mRNA in both the BMDMs and the liver tissues and the phosphorylation of IRF3 and STING proteins in the BMDMs.Conclusion LXJDHYF can significantly improve liver function and attenuate inflammation in ACLF mice possibly by inhibiting excessive activation of the cGAS-STING signaling pathway.

OBJECTIVES: To explore the role of the cGAS-STING signaling pathway in the therapeutic mechanism of Liangxue Jiedu Huayu Formula (LXJDHYF) for acute-on-chronic liver failure (ACLF) in mice. METHODS: Thirty C57BL/6 mice were randomly divided into blank control group, model group, low- and high-dose LXJDHYF groups, and H151 (a specific cGAS-STING pathway inhibitor) group (n=6). In all but the control group, the mice were treated with CCl₄ to induce liver cirrhosis followed by intraperitoneal injections of lipopolysaccharide and D-amino galactose to establish mouse models of ACLF. After the treatments, the mouse livers were collected for HE and TUNEL staining, and serum levels of ALT, AST and TBil were determined. In bone marrow-derived macrophages (BMDMs) and liver tissues of ACLF mice, the expressions of cGAS-STING signaling pathway-related mRNAs including IFN‑β, ISG15, IL-6 and TNF-α were determined with RT-qPCR, and the phosphorylation levels of IRF3 and STING proteins were investigated using Western blotting. RESULTS: Compared with the mice in the model group, the LXJDHYF-treated mice exhibited milder hepatocyte necrosis and inflammatory cell infiltration in the liver with significantly reduced hepatocyte apoptosis. LXJDHYF treatment also significantly lowered serum levels of ALT, AST, TBil, IL-6 and TNF-α in ACLF mice and effectively suppressed the expressions of cGAS-STING signaling pathway-related mRNA in both the BMDMs and the liver tissues and the phosphorylation of IRF3 and STING proteins in the BMDMs. CONCLUSIONS LXJDHYF can significantly improve liver function and attenuate inflammation in ACLF mice possibly by inhibiting excessive activation of the cGAS-STING signaling pathway.
Animals ; Signal Transduction/drug effects* ; Mice ; Nucleotidyltransferases/metabolism* ; Mice, Inbred C57BL ; Acute-On-Chronic Liver Failure/etiology* ; Membrane Proteins/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Liver/metabolism* ; Disease Models, Animal ; Interferon Regulatory Factor-3/metabolism* ; Interleukin-6/metabolism* ; Male

Objective:In this study,three methods of removing microglial cells from primary astrocyte culture were compared and analyzed to explore the characteristics of different purification methods.It provides the basis for research-ers to select the appropriate primary astrocyte culture method.Methods:The cerebral cortex cells of SD neonatal rats were isolated and platted and divided into an unpurified group(control group),a traditional oscillation purification group(TO group),a cytosine β-D-arabinofuranoside(AraC)+TO group,and an AraC+L-leucine methyl ester(LME)group according to different purification methods.Cell counts were used to calculate the cell production after different purification treatments.The cell morphology of each group was observed under an optical microscope,and the proportion of glial fibrillary acidic protein(GFAP)positive cells was calculated by immunofluorescence staining.AlamarBlue HS was used to detect cell viability after passage.Results:Compared with the control group,the cell purity(P＜0.0001)and cell viability(P＜0.0001)of purified cells in the TO group were significantly increased after pas-sage,while the total number of purified cells was significantly decreased(P＜0.001).These trends were more obvious in the AraC+TO and AraC+LME group,and the cell purity(P＜0.0001 or P＜0.0001)and cell viability(P＜0.01 or P＜0.001)of purified cells in the AraC+TO and AraC+LME group after passage were significantly higher than those in the TO group.The total amount of cells obtained was significantly decreased compared with the TO group(P＜0.01 orP＜0.01).There was no significant difference between the AraC+TO and AraC+LME group.Conclusion:The culture rich in astrocytes(cell purity＞90％)was obtained by the TO method,and the cell yield was high,which could be applied to routine cell research.The highly enriched astrocyte cultures(cell purity＞99％)obtained by the AraC+TO and AraC+LME purification methods can be applied to study the precise role of astrocytes in the context of neuroinflammation.

Objective:To explore the effect of dyslipidemia on the clinical outcome of intracytoplasmic sperm injection-embryo transfer (ICSI-ET) in infertility patients receiving donor eggs.Methods:A total of 118 patients were selected to receive egg donors and ICSI-ET at the First Affiliated Hospital of Nanjing Medical University between April 2007 and December 2020. According to the levels of triacylglycerol, serum cholesterol, high density lipoprotein (HDL), and low density lipoprotein, they were divided into dyslipidemia group (35 cases) and normal blood lipids group (83 cases). The influence of body mass index (BMI) and age was adjusted by 1∶1 propensity score matching, and the general condition and clinical outcome of the two groups were analyzed retrospectively. Finally, the relationship between lipid composition and clinical outcome was analyzed according to patients′ age and BMI.Results:(1) Comparing the pre-matching dyslipidemia group with the normal blood lipids group, the BMI of the dyslipidemia group was significantly higher than that of the normal blood lipids group [(23.5±2.4) vs (22.4±2.7) kg/m 2], and the embryo implantation rate was significantly lower than that of the normal blood lipids group [13.6% (8/59) vs 27.3% (36/132)], the differences were statistically significant (both P<0.05). (2) There were no significant differences in years of infertility, number of pregnancies, number of abortions, number of transplanted embryos, protocol of endometrial preparation, endometrial thickness on transplantation day and high quality embryo rate between the two groups, through propensity score matching (all P>0.05). The biochemical pregnancy rate [28.6% (10/35)], embryo implantation rate [13.6% (8/59)] and live birth rate [20.0% (7/35)] in dyslipidemia group were significantly lower than those in the normal blood lipids group ( P<0.05). The clinical pregnancy rate was lower than that of the normal blood lipids group ( P>0.05). (3) The results of stratified analysis showed that the level of HDL in the clinically non-pregnant group was significantly lower than that in the pregnant group in patients ≤ 35 years old [(1.5±0.3) vs (1.8±0.5) mmol/L; P<0.05]. In the overweight recipient patients, the level of HDL of the clinically non-pregnant group was lower than that of the pregnant group ( P>0.05). Conclusions:Dyslipidemia significantly reduces the biochemical pregnancy rate, embryo implantation rate and live birth rate in patients with receiving donor eggs. Especially in patients aged ≤35 years old, the reduction of HDL is closely related to adverse pregnancy outcomes.

Objective To explore the relationship between renal volume and function in the preterm and term newborns at different gestational ages.Methods This study was conducted at the First Affiliated Hospital of Zhengzhou University from October 2016 to March 2018.A total of 626 newborns with different gestational ages were included and the renal volume was determined by ultrasonography.Blood samples were taken for laboratory tests to detect renal function including urea,creatinine,uric acid and estimated glomerular filtration rate.Results A total of 352 preterm and 274 full term newborns were enrolled at birth.The mean gestational age of the neonates was (36.0 ± 3.5) weeks and the mean birth weight was (2.59 ±0.77) kg.The mean renal volume of the preterm infants was (19.57 ±4.30) cm3 and estimated glomerular filtration was (21.68 ± 5.99) mL/(min · 1.73 m2);the mean renal volume of the term infants was(23.03 ± 3.80) cm3 and estimated glomerular filtration was(46.60 ± 10.21) mL/(min · 1.73 m2).The renal volume and estimated glomerular filtration of term infants was significantly greater compared to the preterm infants (t =12.96,33.10,all P ＜ 0.001).The renal volume was highly linear positively correlated with gestational age,birth weight and birth length (r =0.546,0.605,0.592,all P ＜ 0.001).The renal volume was highly linear positively correlated with estimated glomerular filtration(r =0.396,P ＜ 0.001).The renal volume was negatively correlated with urea,creatinine and uric acid(r =-0.210,-0.280,-0.176,all P ＜ 0.001).Conclusions The renal volume increases with gestational age and birth weight in neonates.Estimated glomerular filtration increases with renal volume in neonates.The preterm infants have immaturity kidney size and poor development so that they need special medical care.

Objective To explore the testis development and epididymis in the preterm and term newborns so as to provide the scientific evidence for early clinical diagnosing early.Methods From October 2016 to March 2018,456 hospitalized neonates at Department of Neonatology,the First Affiliated Hospital of Zhengzhou University were recruited within 7 days at birth in this study.In these patients,224 cases were preterm newborns and 232 cases were term newborns.These gestational ages of newborns at birth were (36.18 ± 3.13) weeks (27-41 +6 weeks) and weighted (2.66 ± 0.67) kg(0.90-3.82 kg).The size of the testis and epididymis were measured by ultrasonography.Results The mean testicular volume of the preterm was (0.24 ± 0.07) mL.The mean length of epididymal head,thickness of epididymal head and body and tail of the preterm newborns were (4.17 ±0.59) mm,(2.58 ±0.39) mm,(1.78 ± 0.26) mm,(1.91 ± 0.24) mm,respectively.The mean testicular volume of the term newborns was (0.38 ± 0.13) mL,the mean length of epididymal head,thickness of epididymal head and body and tail of the term newborns were (4.49 ± 0.45) mm,(2.78 ± 0.34) mm,(1.95 ± 0.20) mm,(1.99 ± 0.16) mm,respectively.The mean testicular volume,length of epididymal head,thickness of epididymal head and body and tail of the preterm newborns were significantly lower compared with the term newborns (t =12.810,8.261,6.819,8.058,3.591,all P ＜0.001).The mean testicular volume of the newborns were highly linear positively correlated with gestational age,birth weight and birth length (r =0.538,0.591,0.533,all P ＜ 0.001).In the preterm newborns at postmenstrual age (PMA) of 37 weeks,the mean testicular volume,length of epididymal head,thickness of epididymal head and body and tail of the preterm newborns had no significant difference between the 2 groups (t =1.561,0.863,0.282,1.732,1.147,all P ＞ 0.05).Conclusions The testicular volume,the length of epididymal head,thickness of epididymal head and body increase with gestational age,birth weight and birth length in early neonates.The growth of reproductive system in the preterm newborns at PMA 37 weeks catch-up with term newborns.If this catch-up growth was incomplete at PMA 37 weeks,special attention should be given to monitor underlying diseases.