ObjectiveTo study the effect of Zuoguiwan on the development of skin barrier in the neonatal rat model of congenital kidney deficiency and unveil the underlying mechanism. MethodsSixty rats were paired in a female-to-male ratio of 2∶1， and the pregnant rats were assigned into control， congenital kidney deficiency， and low- and high-dose （2 and 8 g·kg^-1， respectively） Zuoguiwan groups. The pregnant rats in other groups except the control group were exposed to chronic unpredictable mild stress for the modeling of congenital kidney deficiency. The rats in the control group and congenital kidney deficiency group were administrated with normal saline by gavage， and those in Zuoguiwan groups with Zuoguiwan suspension by gavage from day 1 of pregnancy. The serum level of interleukin-6 （IL-6） in the neonatal rats on the day of birth was determined by enzyme-linked immunosorbent assay. The full-thickness skin of neonatal rats on the day of birth was removed from the same position on the back and stained with hematoxylin-eosin for observation of histopathological changes， measurement of skin thickness， and counting of hair follicles. Terminal deoxynucleotidyl transferase-mediated nick end labeling was used to detect the apoptosis of skin tissue cells. The expression of interleukin-6 receptor （IL-6R） and interleukin-17A （IL-17A） in the skin tissue was assessed by immunohistochemistry and Western blot， and the expression of occludin， connexin 43 （Cx43）， and zonula occludens-1 （ZO-1） in the skin tissue was assessed by immunofluorescence and Western blot. ResultsCompared with those in the control group， the neonatal rats in the congenital kidney deficiency group showed a rise in the serum IL-6 level （P<0.01）， decreases in stratum corneum thickness， skin thickness， and number of hair follicles （P<0.01）， increases in the expression of IL-6R and IL-17A in the skin tissue （P<0.01） and the number of apoptotic cells （P<0.01）， and decreases in the expression of occludin， Cx43， ZO-1 （P<0.05）. Compared with the congenital kidney deficiency group， the low- and high-dose Zuoguiwan groups showed declines in serum IL-6 level （P<0.05）. The low-dose group showed increased number of hair follicles （P<0.05）， and the high-dose group presented thickened stratum corneum （P<0.01）， increased number of hair follicles （P<0.01）， and down-regulated expression of IL-6R and IL-17A in the skin tissue （P<0.05， P<0.01）. Both Zuoguiwan groups showcased decreased number of apoptotic cells （P<0.05， P<0.01）， and the high-dose group showed up-regulated expression of occludin， Cx43， and ZO-1 in the skin tissue （P<0.05， P<0.01）. ConclusionZuoguiwan can reduce the levels of IL-6 in the serum and IL-6R and IL-17A in the skin tissue and improve the expression of intercellular junction proteins， thereby ameliorating the abnormal development of the skin barrier in the neonatal rat model of congenital kidney deficiency.

ObjectiveTo study the effect of Zuoguiwan on the development of skin barrier in the neonatal rat model of congenital kidney deficiency and unveil the underlying mechanism. MethodsSixty rats were paired in a female-to-male ratio of 2∶1， and the pregnant rats were assigned into control， congenital kidney deficiency， and low- and high-dose （2 and 8 g·kg^-1， respectively） Zuoguiwan groups. The pregnant rats in other groups except the control group were exposed to chronic unpredictable mild stress for the modeling of congenital kidney deficiency. The rats in the control group and congenital kidney deficiency group were administrated with normal saline by gavage， and those in Zuoguiwan groups with Zuoguiwan suspension by gavage from day 1 of pregnancy. The serum level of interleukin-6 （IL-6） in the neonatal rats on the day of birth was determined by enzyme-linked immunosorbent assay. The full-thickness skin of neonatal rats on the day of birth was removed from the same position on the back and stained with hematoxylin-eosin for observation of histopathological changes， measurement of skin thickness， and counting of hair follicles. Terminal deoxynucleotidyl transferase-mediated nick end labeling was used to detect the apoptosis of skin tissue cells. The expression of interleukin-6 receptor （IL-6R） and interleukin-17A （IL-17A） in the skin tissue was assessed by immunohistochemistry and Western blot， and the expression of occludin， connexin 43 （Cx43）， and zonula occludens-1 （ZO-1） in the skin tissue was assessed by immunofluorescence and Western blot. ResultsCompared with those in the control group， the neonatal rats in the congenital kidney deficiency group showed a rise in the serum IL-6 level （P<0.01）， decreases in stratum corneum thickness， skin thickness， and number of hair follicles （P<0.01）， increases in the expression of IL-6R and IL-17A in the skin tissue （P<0.01） and the number of apoptotic cells （P<0.01）， and decreases in the expression of occludin， Cx43， ZO-1 （P<0.05）. Compared with the congenital kidney deficiency group， the low- and high-dose Zuoguiwan groups showed declines in serum IL-6 level （P<0.05）. The low-dose group showed increased number of hair follicles （P<0.05）， and the high-dose group presented thickened stratum corneum （P<0.01）， increased number of hair follicles （P<0.01）， and down-regulated expression of IL-6R and IL-17A in the skin tissue （P<0.05， P<0.01）. Both Zuoguiwan groups showcased decreased number of apoptotic cells （P<0.05， P<0.01）， and the high-dose group showed up-regulated expression of occludin， Cx43， and ZO-1 in the skin tissue （P<0.05， P<0.01）. ConclusionZuoguiwan can reduce the levels of IL-6 in the serum and IL-6R and IL-17A in the skin tissue and improve the expression of intercellular junction proteins， thereby ameliorating the abnormal development of the skin barrier in the neonatal rat model of congenital kidney deficiency.

ObjectiveThe active ingredients， action targets， and signaling pathways of Cuscutae Semen to control premature ovarian failure were initially predicted by network pharmacology and molecular docking techniques， and an animal model of premature ovarian failure was constructed to explore the mechanism of Cuscutae Semen based on lipid and atherosclerosis signaling pathways. MethodThe effective components and corresponding targets of drugs were obtained from Traditional Chinese Medicines Systems Pharmacology Platform （TCMSP）， Swiss Target Prediction， Pharmmapper， and other databases. GeneCards database was used to collect disease-related targets. Venny2.1.0 online tool was used to screen out the intersection targets of drugs and diseases， and STRING database and Cytoscape v3.7.2 software were used to construct the network diagram of "drug-component-target" and protein-protein interaction （PPI）. The gene ontology （GO） and the Kyoto encyclopedia of genes and genomes （KEGG） enrichment analyses of the intersection targets were performed by running the R language script. The molecular docking technology was utilized to dock drug components with targets and visualize some of the docking results. The mice were randomly divided into a blank group， a model group， a Cuscutae Semen group， and an estradiol valerate group， and the ovarian premature failure model was prepared by chronic stress. The blank group and the model group were gavaged with the same amount of normal saline， and the Cuscutae Semen group was given a Cuscutae Semen decoction of 2.6 g·kg^-1·d^-1. The estradiol valerate group was given an estradiol valerate solution of 0.13 mg·kg^-1·d^-1. After four weeks， samples were collected， and hematoxylin-eosin （HE） staining was performed to observe the histopathological changes in the ovary. Serum levels of follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， estradiol （E₂）， Muller's tube inhibitor/anti-Muller's tube hormone （AMH）， total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， and low-density lipoprotein cholesterol （LDL-C） were determined by enzyme-linked immunosorbent assay （ELISA）. The expression levels of extracellular regulatory protein kinase （ERK）， nuclear transcription factor-κB p65 （NF-κB p65）， nuclear transcription factor-κB suppressor α （IκBα）， interleukin-1β （IL-1β）， IL-6， and tumor necrosis factor-α （TNF-α） were measured by Western blot. ResultA total of 171 targets of Cuscutae Semen for the prevention and treatment of premature ovarian failure were screened， mainly including tumor protein p53 （TP53）， protein kinase B1 （Akt1）， sarcoma （SRC）， tumor necrosis factor （TNF）， epidermal growth factor receptor （EGFR）， etc. KEGG pathway enrichment analysis predicts that Cuscutae Semen is mainly involved in lipid and atherosclerosis， TNF signaling pathway， and TP53 signaling pathway to control premature ovarian failure. The animal experiments show that compared with the premature ovarian failure model group， the Cuscutae Semen group can significantly upregulate AMH， E₂， and HDL-C （P<0.05， P<0.01）， significantly downregulate LH， TC， and LDL-C （P<0.01）， greatly reduce IL-1β， IL-6， and TNF-α protein levels， as well as ERK， NF-κB p65， and their phosphorylation levels （P<0.01）. ConclusionCuscutae Semen can regulate hormone levels and improve ovarian function through a multi-component， multi-target， and multi-pathway approach， and the mechanism may be related to the regulation of lipid and atherosclerosis signaling pathways.

ObjectiveTo observe the effect of Zuoguiwan on ovarian reserve in the female offspring rat model of prenatal stress （PS） and explore the mechanism based on Toll-like receptor 4/nuclear factor-κB p65 （TLR4/NF-κB p65） signaling pathway. MethodThirty-two pregnant rats were prepared and randomized into four groups （n=8）： control， model， Zuoguiwan （18.9 mg·kg^-1）， and vitamin E （1.44 mg·kg^-1）. Except the control group， the other three groups were subjected to chronic unpredictable mild stress （CUMS） from day 11 of pregnancy， and the modeling was accompanied by gavage with corresponding drugs until delivery. The PS model was evaluated by the sucrose preference test， open field test， and serum corticosterone （CORT） level. The estrous cycle was monitored and the morphological changes in the ovarian tissue were observed. The serum levels of estradiol （E₂）， luteinizing hormone （LH）， follicle-stimulating hormone （FSH）， and anti-Mullerian hormone （AMH） in the 75-day-old offspring rats were measured by enzyme-linked immunosorbent assay （ELISA） to evaluate the ovarian reserve. The ovary and uterus indices were calculated. The serum levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） were measured by enzyme-linked immunosorbent assay （ELISA）. The morphology of the ovarian tissue in the offspring on the day of birth and day 75 after birth was observed by hematoxylin-eosin staining. The transport of NF-κB p65 to the nucleus in the ovaries of the 75-day-old offspring was detected by the immunofluorescence （IF） assay. The expression of TLR4， NF-κB p65 and other related proteins in the ovarian tissue was determined by Western blot. ResultCompared with the control group， the model group showed reduced primordial follicles in the offspring on the day of birth （P<0.01） as well as disturbed estrous cycle， decreased ovary index and uterus index （P<0.01）， reduced corpus luteum， increased atretic follicles （P<0.01）， lowered serum levels of AMH and E₂ （P<0.01）， elevated serum levels of LH， FSH， IL-1β， and TNF-α （P<0.05， P<0.01）， and up-regulated protein levels of TLR4， NF-κB p65， recombinant myeloid differentiation factor 88 （MyD88）， and phosphorylated NF-κB inhibitor （p-IκBα） （P<0.01） in the 75-day-old offspring rats. Compared with the model group， Zuoguiwan and vitamin E increased the primordial follicles in the offspring on the day of birth （P<0.01）. Moreover， they resumed the estrous cycle， increased the ovary and uterine indices （P<0.05， P<0.01） and corpus luteum （P<0.01）， reduced atretic follicles （P<0.01）， elevated the serum levels of AMH and E₂ （P<0.05， P<0.01）， lowered the serum levels of LH， FSH， IL-1β， and TNF-α （P<0.05， P<0.01）， and down-regulated the expression of TLR4， NF-κB p65， MyD88， and p-IκB-α （P<0.05， P<0.01） in the 75-day-old offspring. ConclusionZuoguiwan can improve the ovarian reserve in the offspring rat model of congenital kidney deficiency by regulating the TLR4/NF-κB p65 signaling pathway.

Objective:To establish a general method for the simultaneous determination of hydroxyphenyl esters and quaternary ammonium bacteriostatic agents in eye drops.Methods:The chromatographic analysis was per-formed on an Agilent C18 column(4.6 mm ×250 mm,5 μm)with 1％triethylamine solution(pH adjusted to 5.0 with phosphoric acid)as mobile phase A and methanol as mobile phase B.Gradient elution was performed at col-umn temperature of 40 ℃.The detection wavelength was 214 nm,the flow rate was 1 mL·min-1,and the injec-tion volume was 20 μL.Results:Methylparaben,ethylparaben,propylparaben,butylparaben,benzalkonium chlo-ride and benzalkonium bromide were 0.11-559.0,0.10-513.0,0.10-258.8,0.11-270.5,1.07-537.0 and 1.03-512.8 μg·mL-1,respectively.The linear range was good(r＞0.999).The average recoveries of meth-ylparaben,benzalkonium bromide and benzalkonium chloride were 104.7％(RSD=1.3％),102.6％(RSD=1.1％)and 100.9％(RSD=1.1％),respectively.The contents of bacteriostatic agent in 100 batches of eye drops from 36 varieties of 12 enterprises were determined,and the accurate results were obtained.Conclusion:This meth-od provides a reference for the content quality control and safety evaluation of bacteriostatic agents in eye drops.

ObjectiveTo explore the therapeutic effect and mechanism of intermittent theta burst transcranial magnetic stimulation (iTBS) on non-fluent aphasia after stroke. MethodsFrom August, 2021 to August, 2022, 50 patients with non-fluent aphasia after stroke in the First People's Hospital of Lianyungang were randomly divided into sham stimulation group (n = 25) and iTBS group (n = 25). Both groups accepted speach training. iTBS group accepted iTBS, and the sham stimulation group received sham iTBS, for four weeks. The serum brain-derived neurotrophic factor (BDNF) was measured, and they were assessed by China Rehabilitation Research Center Standard Aphasia Examination (CRRCAE) and Boston Diagnostic Aphasia Examination before and after treatment. ResultsTwo cases in the sham stimulation group and three cases in iTBS group dropped down. The BDNF level, and listen and understand, repeat, name, read aloud, and total score of CRRCAE improved in two groups after treatment (|t| > 5.012, P < 0.001); and they were better in iTBS group than in the sham stimulation group (|t| > 3.968, P < 0.001). The total effective rate was more in iTBS group than in the sham stimulation group (χ² = 8.835, P < 0.05). ConclusioniTBS can improve speech function in patients with non-fluent aphasia after stroke, which may associate with the promotion of BDNF.

Self-organized blastoids from extended pluripotent stem (EPS) cells possess enormous potential for investigating postimplantation embryo development and related diseases. However, the limited ability of postimplantation development of EPS-blastoids hinders its further application. In this study, single-cell transcriptomic analysis indicated that the "trophectoderm (TE)-like structure" of EPS-blastoids was primarily composed of primitive endoderm (PrE)-related cells instead of TE-related cells. We further identified PrE-like cells in EPS cell culture that contribute to the blastoid formation with TE-like structure. Inhibition of PrE cell differentiation by inhibiting MEK signaling or knockout of Gata6 in EPS cells markedly suppressed EPS-blastoid formation. Furthermore, we demonstrated that blastocyst-like structures reconstituted by combining the EPS-derived bilineage embryo-like structure (BLES) with either tetraploid embryos or tetraploid TE cells could implant normally and develop into live fetuses. In summary, our study reveals that TE improvement is critical for constructing a functional embryo using stem cells in vitro.
Pregnancy ; Female ; Animals ; Mice ; Tetraploidy ; Blastocyst ; Embryo, Mammalian ; Cell Differentiation ; Embryonic Development

Objective:To investigate the short-term effects of acute fructose intake on serum antioxidant capacity and liver enzymes in healthy young adults.Methods:From January to June 2019, 64 healthy young subjects were recruited, and divided into 75 g glucose group, 25 g fructose group, 50 g fructose group and 75 g fructose group by random digits table method with 16 cases each. The subjects took corresponding amounts of glucose or fructose according to grouping. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), glutathione peroxidase (GPX), superoxide dismutase (SOD), C-Jun amino terminal kinase (JNK), malondialdehyde (MDA) and 8-OH deoxyguanine (8-OHdG) before taking sugar and 30, 60, 120, 180 min after taking sugar, and the changes of ALT, AST and LDH at 30, 60, 120 and 180 min after taking sugar compared with that before taking sugar.Results:One case in 50 g fructose group, 2 cases in 75 g fructose group and 1 case in 75 g glucose group dropped out due to adverse reaction; finally, 15 cases in 75 g glucose group, 16 cases in 25 g fructose group, 15 cases in 50 g fructose group and 14 cases in 75 g fructose group completed the study. The increase of ALT and AST after taking sugar in 25 g fructose group, 50 g fructose group and 75 g fructose group was significantly higher than that in 75 g glucose group, and there were statistical differences ( P<0.05); there was no statistical difference in the change of LDH after taking sugar among 4 groups ( P>0.05). One hundred and eighty min after taking sugar, the receiver operating characteristic (ROC) curve analysis result showed that there were no statistical differences in the areas under curve of ALT, AST and LDH among 4 groups ( P>0.05). There was no statistical difference in SOD before taking sugar among 4 groups ( P>0.05); the SOD 60 min after taking sugar in 50 g fructose group and 75 g fructose group, and SOD 180 min after taking sugar in 25 g fructose group, 50 g fructose group and 75 g fructose group were significantly lower than those in 75 g glucose group: (4.84 ± 1.88) and (4.38 ± 1.12) μg/L vs. (6.25 ± 1.65) μg/L, (4.46 ± 1.66), (5.22 ± 1.66) and (3.99 ± 0.96) μg/L vs. (6.55 ± 1.78) μg/L, and there were statistical differences ( P<0.05). There were no statistical differences in the changes of JNK, GPX, MDA and 8-OHdG before and after taking sugar among 4 groups ( P>0.05). The ROC curve 180 min after taking sugar analysis result showed that the area under curve of SOD in 75 g fructose group was significantly lower than that in 75 g glucose group (9.06 ± 1.88 vs. 12.74 ± 3.15), and there was statistical difference ( P<0.05); there were no statistical differences in the areas under curve of GPX, JNK, MDA and 8-OHdG among 4 groups ( P>0.05). Conclusions:Acute fructose intake can lead to the decrease of antioxidant capacity, and the increasing of oxidative damage and liver enzymes in healthy adults.

As a sensor of cytosolic DNA, the role of cyclic GMP-AMP synthase (cGAS) in innate immune response is well established, yet how its functions in different biological conditions remain to be elucidated. Here, we identify cGAS as an essential regulator in inhibiting mitotic DNA double-strand break (DSB) repair and protecting short telomeres from end-to-end fusion independent of the canonical cGAS-STING pathway. cGAS associates with telomeric/subtelomeric DNA during mitosis when TRF1/TRF2/POT1 are deficient on telomeres. Depletion of cGAS leads to mitotic chromosome end-to-end fusions predominantly occurring between short telomeres. Mechanistically, cGAS interacts with CDK1 and positions them to chromosome ends. Thus, CDK1 inhibits mitotic non-homologous end joining (NHEJ) by blocking the recruitment of RNF8. cGAS-deficient human primary cells are defective in entering replicative senescence and display chromosome end-to-end fusions, genome instability and prolonged growth arrest. Altogether, cGAS safeguards genome stability by controlling mitotic DSB repair to inhibit mitotic chromosome end-to-end fusions, thus facilitating replicative senescence.

Objective:To investigate the acute effect of fructose intake on serum uric acid, plasma glucose, and insulin levels in healthy young adults.Methods:Sixty-four healthy young subjects were recruited, and randomized to 25 g, 50 g, 75 g fructose group, and 75 g glucose group( n=16) by random number table. The anthropometric parameters, blood pressure, heart rate were measured. Several biochemistry parameters were measured, which were serum uric acid, plasma glucose, plasma insulin, serum total cholesterol, triglyceride, low density lipoprotein-cholesterol (LDL-C) at 0, 60, 120, and 180 min before and after ingestion of fructose or glucose. Results:(1) The serum uric acid level after fructose administration increased significantly than after glucose over 3 h, and peaked at 60 min. The increment of uric acid at 60 min and area under curve of uric acid at 3 h after fructose administration were significantly higher than those of glucose. The increment of uric acid at 60 min increased significantly as fructose dose was increased, especially in the 75 g fructose (increment rate of uric acid at 60 min in 25g, 50g, 75g fructose groups were 9.33%, 13.11%, 17.69% vs 0.75% respectively; Areas under curve of uric acid were 1 674.1±410.38, 1 598.3±417.03, 1 504.6±292.46 vs 1 434.8±328.94, P<0.01). (2) The glucose and insulin levels increased after fructose/glucose intake in four groups with top augment in glucose followed by 75 g fructose. The increase peaked at 30 min, began to decline at 120 min, and returned to fasting level at 180 min. The area under curve of insulin at glucose group was significantly higher than those among fructose groups. With the increase of fructose dose, the increment rate of glucose and insulin at 60 min also increased obviously, especially in the 75 g fructose (the increment rates of glucose at 60 min in 25 g, 50 g, 75 g fructose, 75 g glucose were 7.40%, 8.29%, 13.74%, 28.22% respectively; The increment rates of insulin at 60 min were 54.29%, 115.25%, 185.58%, 730.31% respectively, P<0.01). (3) There were no difference of cholesterol, triglyceride, and LDL-C after fructose/glucose ingestion. Conclusion:Acute fructose intake can lead to the increase of uric acid and insulin; Moreover, the increments of uric acid and insulin after fructose consumption were dependent on fructose dose.