Aim To explore the effect of astragalosideⅣ(AS-Ⅳ)on lipopolysaccharide(LPS)-induced po-larization and migration of RAW 264.7 macrophages and the underlying mechanism.Methods 1 mg·L-1 LPS was used to construct cell migration model.Scratch assay was utilized to determine cell migration rate.Immunofluorescence staining was utilized to de-tect the expression and location of F4/80,iNOS and Arg-1.CCK-8 assay was used to determine the viabili-ty of RAW 264.7 cells.Griess assay was used to measure NO content.Molecular docking was used to analyze the interaction between AS-Ⅳ and the core tar-gets such as cGAS and STING protein.Western blot was employed to detect the expression of iNOS,Arg-1,cGAS,STING,NF-κB p65 and p-NF-κB p65 protein.Results AS-Ⅳ significantly inhibited the migration and M1 polarization of RAW 264.7 cells induced by LPS.Moreover,AS-Ⅳ could interact with cGAS and STING protein,especially cGAS.Further Western blot assay showed that AS-Ⅳ significantly downregulated the expression of iNOS,cGAS,STING and p-NF-κB p65 protein.Conclusions AS-Ⅳ could promote mac-rophage M1 to M2 polarization,thereby inhibited mac-rophage migration through restraining the cGAS/STING/NF-κB signaling pathway,which provides a new therapeutic target for AS-Ⅳ to improve the early inflammatory response of AS.

Aim To investigate the neuroprotective effect of Takeda G protein-coupled receptor-5(TGR5)activated by INT-777 on hypoxic-ischemic encephalop-athy(HIE)in neonatal rats.Methods Seven-day-old SD rats were randomly divided into the sham opera-tion group(Sham,S),model group(HIE,G),INT-777 low-dose(L),medium-dose(M),and high-dose(H)groups.The modified Rice-Vanucci method was used to construct the HIE model and Intranasal admin-istration 1 h after modeling.Short-term neurobehavioral tests were performed 48 h after modeling to evaluate the neurological function of neonatal rats,TTC staining was used to determine the volume of cerebral infarction,dry and wet specific gravity was used to determine the brain water content,ferrous ion kit was used to deter-mine the brain ferrous ion content,HE staining was used to observe the pathological damage of brain tis-sue,Nissl staining was used to observe the loss of Nissl substance,Transmission electron microscopy(TEM)was used to observe the mitochondrial morphological changes of cortical neurons,and Western blot was em-ployed to detect the expression of ferroptosis-related proteins TFR1 and GPX4.Results Compared with group S,group G had increased short-term neurobehav-ioral test consumption time,higher scores,increased cerebral infarct volume,brain water content,and brain ferrous iron content,significant brain tissue damage on the affected side,severe loss of Nissl substance,smaller neuronal mitochondria,decreased mitochondrial cris-tae,and increased expression of TFR1 and reduced ex-pression of GPX4.Compared with group G,the INT-777 administration group had a shorter consumption time for short-term neurobehavioral tests,lower scores,the cerebral infarction volume,brain water content,and brain ferrous ion content decreased,the brain tissue damage on the affected side was reduced,and there was insignificant loss of Nissl substance,larger neuronal mi-tochondrial volume,increased mitochondrial cristae,re-duced expression of TFR1,and increased expression of GPX4.Conclusions INT-777 agonist TGR5 has a protective effect against hypoxic-ischemic encephalopa-thy in neonatal rats,and its mechanism of action may be related to the inhibition of neuronal ferroptosis.

Aim To explore the effect of astragalosideⅣ(AS-Ⅳ)on lipopolysaccharide(LPS)-induced po-larization and migration of RAW 264.7 macrophages and the underlying mechanism.Methods 1 mg·L-1 LPS was used to construct cell migration model.Scratch assay was utilized to determine cell migration rate.Immunofluorescence staining was utilized to de-tect the expression and location of F4/80,iNOS and Arg-1.CCK-8 assay was used to determine the viabili-ty of RAW 264.7 cells.Griess assay was used to measure NO content.Molecular docking was used to analyze the interaction between AS-Ⅳ and the core tar-gets such as cGAS and STING protein.Western blot was employed to detect the expression of iNOS,Arg-1,cGAS,STING,NF-κB p65 and p-NF-κB p65 protein.Results AS-Ⅳ significantly inhibited the migration and M1 polarization of RAW 264.7 cells induced by LPS.Moreover,AS-Ⅳ could interact with cGAS and STING protein,especially cGAS.Further Western blot assay showed that AS-Ⅳ significantly downregulated the expression of iNOS,cGAS,STING and p-NF-κB p65 protein.Conclusions AS-Ⅳ could promote mac-rophage M1 to M2 polarization,thereby inhibited mac-rophage migration through restraining the cGAS/STING/NF-κB signaling pathway,which provides a new therapeutic target for AS-Ⅳ to improve the early inflammatory response of AS.

Aim To investigate the neuroprotective effect of Takeda G protein-coupled receptor-5(TGR5)activated by INT-777 on hypoxic-ischemic encephalop-athy(HIE)in neonatal rats.Methods Seven-day-old SD rats were randomly divided into the sham opera-tion group(Sham,S),model group(HIE,G),INT-777 low-dose(L),medium-dose(M),and high-dose(H)groups.The modified Rice-Vanucci method was used to construct the HIE model and Intranasal admin-istration 1 h after modeling.Short-term neurobehavioral tests were performed 48 h after modeling to evaluate the neurological function of neonatal rats,TTC staining was used to determine the volume of cerebral infarction,dry and wet specific gravity was used to determine the brain water content,ferrous ion kit was used to deter-mine the brain ferrous ion content,HE staining was used to observe the pathological damage of brain tis-sue,Nissl staining was used to observe the loss of Nissl substance,Transmission electron microscopy(TEM)was used to observe the mitochondrial morphological changes of cortical neurons,and Western blot was em-ployed to detect the expression of ferroptosis-related proteins TFR1 and GPX4.Results Compared with group S,group G had increased short-term neurobehav-ioral test consumption time,higher scores,increased cerebral infarct volume,brain water content,and brain ferrous iron content,significant brain tissue damage on the affected side,severe loss of Nissl substance,smaller neuronal mitochondria,decreased mitochondrial cris-tae,and increased expression of TFR1 and reduced ex-pression of GPX4.Compared with group G,the INT-777 administration group had a shorter consumption time for short-term neurobehavioral tests,lower scores,the cerebral infarction volume,brain water content,and brain ferrous ion content decreased,the brain tissue damage on the affected side was reduced,and there was insignificant loss of Nissl substance,larger neuronal mi-tochondrial volume,increased mitochondrial cristae,re-duced expression of TFR1,and increased expression of GPX4.Conclusions INT-777 agonist TGR5 has a protective effect against hypoxic-ischemic encephalopa-thy in neonatal rats,and its mechanism of action may be related to the inhibition of neuronal ferroptosis.

Isoflavones which mainly distributed in leguminous plants have plenty of health benefits.Isoflavone synthase(IFS)is a membrane-associated cytochrome P450 enzyme(CYP450)which carries out the unique aryl-ring migration and hydroxylation.So far,few crystal structures of plant P450s have been obtained.We determined the crystal structure of IFS from Medicago truncatula at 1.9 ? by MAD method using a selenomethionine substituted crystal and conducted molecular docking and mutagenesis study.The structure of IFS complexed with imidazole exhibits the helix Ⅰa-loop-helix Ⅰβ motif which cor-responds to helix Ⅰ of other P450s.Compared with structures of common P450s,IFS/imidazole structure contains an extra domain,i.e.,the γ-domain.The structure reveals a homodimer in which the γ-domain of one molecule interacts with the β-domain of another.The plane of heme group makes an angle of ap-proximately 40° with the helix Ⅰa-loop-helix Ⅰβ motif.Molecular docking combined with mutagenesis study suggested that Trp-128 and Asp-300 might play important roles in substrate binding and recogni-tion.Phe-301,Ser-303 and Gly-305 from the helix Ⅰa-loop-helix Ⅰβ motif may play important roles in the aryl-ring migration.These novel structural features reveal insights into the unique reaction mechanism of IFS and provide a basis for engineering IFS in leguminous crops for health purpose.

Isoflavones which mainly distributed in leguminous plants have plenty of health benefits.Isoflavone synthase(IFS)is a membrane-associated cytochrome P450 enzyme(CYP450)which carries out the unique aryl-ring migration and hydroxylation.So far,few crystal structures of plant P450s have been obtained.We determined the crystal structure of IFS from Medicago truncatula at 1.9 ? by MAD method using a selenomethionine substituted crystal and conducted molecular docking and mutagenesis study.The structure of IFS complexed with imidazole exhibits the helix Ⅰa-loop-helix Ⅰβ motif which cor-responds to helix Ⅰ of other P450s.Compared with structures of common P450s,IFS/imidazole structure contains an extra domain,i.e.,the γ-domain.The structure reveals a homodimer in which the γ-domain of one molecule interacts with the β-domain of another.The plane of heme group makes an angle of ap-proximately 40° with the helix Ⅰa-loop-helix Ⅰβ motif.Molecular docking combined with mutagenesis study suggested that Trp-128 and Asp-300 might play important roles in substrate binding and recogni-tion.Phe-301,Ser-303 and Gly-305 from the helix Ⅰa-loop-helix Ⅰβ motif may play important roles in the aryl-ring migration.These novel structural features reveal insights into the unique reaction mechanism of IFS and provide a basis for engineering IFS in leguminous crops for health purpose.

Objective::To analyze fetal renal abnormality genetic features and the prenatal characteristics of the 17q12 microdeletion syndrome.Methods::This prospective cohort study examined prenatal ultrasound findings of renal abnormalities in pregnant women who underwent single nucleotide polymorphism (SNP) array or copy number variation sequencing (CNV-seq) testing on amniotic fluid or fetal tissue at Tianjin Central Obstetrics and Gynecology Hospital between January 2016 and August 2022. The study cohort comprised women with advanced maternal age, fetal ultrasound anomalies, high-risk non-invasive prenatal testing results, or suspected 17q12 microdeletion syndrome. Comprehensive clinical data, including maternal age, detailed ultrasound findings, and pregnancy outcomes, were systematically collected. SNP-array analysis was conducted using an Affymetrix CytoScan 750 K Array Chip to identify CNVs and loss of heterozygosity, while CNV-seq was performed on the Illumina HiSeq 2000 platform. Detected variants were classified according to the American College of Medical Genetics and Genomics guidelines. Statistical analyses were performed using SPSS version 27.0.Results::Abnormal renal development was identified in 141 patients, among whom 26 exhibited hyperechogenic kidneys (HCK). Of these, 12 cases were associated with 17q12 microdeletion syndrome, while the remaining 14 were linked to other chromosomal abnormalities. When excluding patients with HCK, those diagnosed with polycystic kidney disease demonstrated a higher prevalence of chromosomal abnormalities compared to those with multicystic dysplastic kidney and renal dysplasia. Although isolated conditions such as horseshoe kidney, hydronephrosis, ectopic kidney, and unilateral kidney typically presented with normal chromosomal findings, the incidence of chromosomal abnormalities increased when these conditions coexisted with other anomalies. A detailed analysis of the correlation between 17q12 microdeletion syndrome and HCK revealed that 12 out of the 14 patients diagnosed with 17q12 microdeletion syndrome exhibited HCK. Genetic testing confirmed the syndrome in seven patients, with five cases attributed to novel mutations and two cases resulting from inherited mutations.Conclusion::Fetal HCK was closely associated with the 17q12 microdeletion syndrome, and polycystic kidney disease showed a higher rate of chromosomal abnormalities. Chromosome test results were mostly normal in patients with other renal abnormalities, such as kidney dysplasia, horseshoe kidneys, hydronephrosis, kidney deficiency, and ectopic kidneys. Prenatal diagnosis is recommended, especially in cases of non-isolated fetal renal abnormalities. This study provides strong evidence supporting a link between fetal renal abnormalities and genetic syndromes.

Objective To establish a technical system for allogeneic kidney transplantation surgery in pigs using the Banna miniature pig inbred strain,and to evaluate it through routine blood tests,liver and kidney function tests,thus providing reference data for the preparation of allogeneic kidney transplantation models.Methods A total of 4 cases of allogeneic kidney transplantation surgeries were performed,including 1 case of single kidney transplantation in a healthy pig,2 cases of kidney transplantation after unilateral nephrectomy,and 1 case of kidney transplantation after bilateral nephrectomy.Before kidney transplantation,cross-matching and complement-dependent cytotoxicity(CDC)tests were used for matching between donor and recipient pigs.After kidney transplantation,peripheral blood samples were regularly collected from pigs for routine blood tests,liver function tests,and kidney function tests,and color Doppler ultrasound technology was used to detect blood supply to the transplanted kidneys.After reaching the experimental endpoint,both kidneys of pig DR1 and the left kidney of pig DR3 were collected and hematoxylin-eosin(HE)staining was performed to evaluate pathological changes in the transplanted kidneys.Results Recipient pigs DR1 and DR3 died at 17 days and 30 days after surgery respectively,while recipient pigs R and DR2 remained in good condition during the 30-day observation period.The results of liver and kidney function test showed that in pig DR1,alanine aminotransferase(ALT)levels increased on postoperative day 1(＞1 000 U/L),peaked on postoperative day 7(1 300 U/L),and aspartate aminotransferase(AST)levels peaked on postoperative day 1(＞3 000 U/L).On postoperative day 17,ALT and AST levels remained high(ALT,500 U/L;AST,700 U/L).In pigs R,DR2,and DR3,ALT and AST levels returned to normal around day 17.Serum creatinine(Crea)levels in pig R remained stable without postoperative increase.Crea levels in pigs DR1 and DR2 showed transient elevation on postoperative day 1,then gradually returned to normal(＜100 μmol/L).Crea levels in pig DR3 remained below 500 μmol/L from postoperative days 2-10,but increased between days 11-28,reaching up to 1 500 μmol/L,indicating gradual loss of kidney function.Ultrasound results showed that the preoperative resistive index(RI)of recipient pig R was 0.91.On postoperative day 24,renal cortex and medulla showed abundant blood flow signals with RI value of 0.88,which was close to the pre-transplantation RI value.For pig DR2,the RI value on postoperative day 17 was 0.89,with poor renal cortex blood flow and relatively good renal medulla blood flow.In pig DR1 on postoperative day 17,no blood flow signals were detected in the transplanted kidney.HE staining results showed that the non-transplanted healthy right kidney of pig DR1 had normal structure,while the transplanted left kidney showed blurred glomerular structure and nuclear dissolution,indicating that the left kidney had lost function before removal.In the transplanted left kidney of pig DR3,large numbers of red blood cells and lymphocyte infiltration were observed in glomeruli and renal tubules,indicating possible coagulation dysfunction and rejection reactions after kidney transplantation.Conclusion Banna miniature pig inbred strain is used as experimental animals to perform four cases of allogeneic pig-to-pig kidney transplantation.The physiological parameters of the recipient pig and the function of the transplanted kidney are monitored after surgery using routine blood tests,liver and kidney function tests,color Doppler ultrasound,and pathological examinations.The allogeneic pig-to-pig kidney transplantation technical system established in the study can provide a foundation for clinicians to conduct kidney transplantation surgeries.

Objective To establish a technical system for allogeneic kidney transplantation surgery in pigs using the Banna miniature pig inbred strain,and to evaluate it through routine blood tests,liver and kidney function tests,thus providing reference data for the preparation of allogeneic kidney transplantation models.Methods A total of 4 cases of allogeneic kidney transplantation surgeries were performed,including 1 case of single kidney transplantation in a healthy pig,2 cases of kidney transplantation after unilateral nephrectomy,and 1 case of kidney transplantation after bilateral nephrectomy.Before kidney transplantation,cross-matching and complement-dependent cytotoxicity(CDC)tests were used for matching between donor and recipient pigs.After kidney transplantation,peripheral blood samples were regularly collected from pigs for routine blood tests,liver function tests,and kidney function tests,and color Doppler ultrasound technology was used to detect blood supply to the transplanted kidneys.After reaching the experimental endpoint,both kidneys of pig DR1 and the left kidney of pig DR3 were collected and hematoxylin-eosin(HE)staining was performed to evaluate pathological changes in the transplanted kidneys.Results Recipient pigs DR1 and DR3 died at 17 days and 30 days after surgery respectively,while recipient pigs R and DR2 remained in good condition during the 30-day observation period.The results of liver and kidney function test showed that in pig DR1,alanine aminotransferase(ALT)levels increased on postoperative day 1(＞1 000 U/L),peaked on postoperative day 7(1 300 U/L),and aspartate aminotransferase(AST)levels peaked on postoperative day 1(＞3 000 U/L).On postoperative day 17,ALT and AST levels remained high(ALT,500 U/L;AST,700 U/L).In pigs R,DR2,and DR3,ALT and AST levels returned to normal around day 17.Serum creatinine(Crea)levels in pig R remained stable without postoperative increase.Crea levels in pigs DR1 and DR2 showed transient elevation on postoperative day 1,then gradually returned to normal(＜100 μmol/L).Crea levels in pig DR3 remained below 500 μmol/L from postoperative days 2-10,but increased between days 11-28,reaching up to 1 500 μmol/L,indicating gradual loss of kidney function.Ultrasound results showed that the preoperative resistive index(RI)of recipient pig R was 0.91.On postoperative day 24,renal cortex and medulla showed abundant blood flow signals with RI value of 0.88,which was close to the pre-transplantation RI value.For pig DR2,the RI value on postoperative day 17 was 0.89,with poor renal cortex blood flow and relatively good renal medulla blood flow.In pig DR1 on postoperative day 17,no blood flow signals were detected in the transplanted kidney.HE staining results showed that the non-transplanted healthy right kidney of pig DR1 had normal structure,while the transplanted left kidney showed blurred glomerular structure and nuclear dissolution,indicating that the left kidney had lost function before removal.In the transplanted left kidney of pig DR3,large numbers of red blood cells and lymphocyte infiltration were observed in glomeruli and renal tubules,indicating possible coagulation dysfunction and rejection reactions after kidney transplantation.Conclusion Banna miniature pig inbred strain is used as experimental animals to perform four cases of allogeneic pig-to-pig kidney transplantation.The physiological parameters of the recipient pig and the function of the transplanted kidney are monitored after surgery using routine blood tests,liver and kidney function tests,color Doppler ultrasound,and pathological examinations.The allogeneic pig-to-pig kidney transplantation technical system established in the study can provide a foundation for clinicians to conduct kidney transplantation surgeries.

Objective To investigate the therapeutic effect of hypericin on acute pancreatitis(AP)in mice and its effect on NLRP3 inflammasome signaling pathway.Methods The AP model in mice was established with caerulein(CER).The mice were divided into the normal control group,the model group(AP group),the low-dose HY group(CER+HY 5 mg/kg group),the medium-dose HY group(CER+HY 10 mg/kg group)and the high-dose HY group(CER+HY 20 mg/kg group),with 10 mice in each group.The 266-6 mouse pancreatic acinar cancer cells were treated with cholecystokinin(CCK)and divided into the control group,the AP group,the CCK+HY 1 μmol/L group,the CCK+HY 2 μmol/L group and the CCK+HY 4 μmol/L group.The activities of amylase(AMS),lipase,trypsin and myeloperoxidase(MPO)in the serum of each group of mice,and levels of inflammatory factors interleukin(IL)-1β and tumor necrosis factor(TNF)-α were detected by enzyme-linked immunosorbent assay(ELISA).The expression of NOD-like receptor family protein 3(NLRP3)was detected by Western blot assay.The mRNA levels of NLRP3,caspase(Caspase)-1,IL-1β,TNF-α and IL-18 in pancreatic tissue of mice were detected by real-time quantitative PCR(q-PCR).The cell survival rate of cells in each group was detected by CCK8 method.The mRNA expression levels of NLRP3,Caspase-1 and IL-18 in each group of cells were detected by q-PCR.Results Compared with the normal control group,the levels of AMS,lipase,MPO,trypsin,IL-1β and TNF-α in serum of the model group,and the mRNA and protein expression levels of NLRP3,IL-1β,TNF-α,IL-18 and Caspase-1 in pancreatic tissue were increased(P＜0.01).Compared with the model group,the levels of AMS,IL-1β and TNF-α,the enzymatic activity of trypsin in serum,and the mRNA levels of IL-1β,TNF-α,IL-18 and Caspase-1 in pancreatic tissue were decreased in the low-,medium-and high-dose HY groups.The serum levels of lipase and MPO and the mRNA expression levels of NLRP3 in pancreatic tissue were decreased in the medium-and high-dose HY groups(P＜0.05).Compared with the AP group,the cell survival rates were increased in the CCK+HY 1 μmol/L group,the CCK+HY 2 μmol/L group and the CCK+HY 4 μmol/L group,and the mRNA levels of NLRP3,IL-18 and Caspase-1 were decreased in a dose-dependent manner(P＜0.05).Conclusion Hypericin can effectively treat AP in vivo and in vitro,and its therapeutic effect may be related to the regulation of NLRP3 inflammasome signaling pathway.