The role of podocyte injury in the pathogenesis of Fabry disease nephropathy / O papel da injúria podocitária na patogênese da nefropatia da doença de Fabry

Abstract Renal involvement is one of the most severe morbidities of Fabry disease (FD), a multisystemic lysosomal storage disease with an X-linked inheritance pattern. It results from pathogenic variants in the GLA gene (Xq22.2), which encodes the production of alpha-galactosidase A (α-Gal), responsible for glycosphingolipid metabolism. Insufficient activity of this lysosomal enzyme generates deposits of unprocessed intermediate substrates, especially globotriaosylceramide (Gb3) and derivatives, triggering cellular injury and subsequently, multiple organ dysfunction, including chronic nephropathy. Kidney injury in FD is classically attributed to Gb3 deposits in renal cells, with podocytes being the main target of the pathological process, in which structural and functional alterations are established early and severely. This configures a typical hereditary metabolic podocytopathy, whose clinical manifestations are proteinuria and progressive renal failure. Although late clinical outcomes and morphological changes are well established in this nephropathy, the molecular mechanisms that trigger and accelerate podocyte injury have not yet been fully elucidated. Podocytes are highly specialized and differentiated cells that cover the outer surface of glomerular capillaries, playing a crucial role in preserving the structure and function of the glomerular filtration barrier. They are frequent targets of injury in many nephropathies. Furthermore, dysfunction and depletion of glomerular podocytes are essential events implicated in the pathogenesis of chronic kidney disease progression. We will review the biology of podocytes and their crucial role in regulating the glomerular filtration barrier, analyzing the main pathogenic pathways involved in podocyte injury, especially related to FD nephropathy.

Resumo O acometimento renal é uma das mais severas morbidades da doença de Fabry (DF), enfermidade multissistêmica de depósito lisossômico com padrão de herança ligada ao cromossomo X, decorrente de variantes patogênicas do gene GLA (Xq22.2), que codifica a produção de alfa-galactosidase A (α-Gal), responsável pelo metabolismo de glicoesfingolipídeos. A atividade insuficiente dessa enzima lisossômica gera depósitos de substratos intermediários não processados, especialmente do globotriaosilceramida (Gb3) e derivados, desencadeando injúria celular e, posteriormente, disfunção de múltiplos órgãos, incluindo a nefropatia crônica. A lesão renal na DF é classicamente atribuída aos depósitos de Gb3 nas células renais, sendo os podócitos o alvo principal do processo patológico, nos quais as alterações estruturais e funcionais são instaladas de forma precoce e severa, configurando uma podocitopatia metabólica hereditária típica, cujas manifestações clínicas são proteinúria e falência renal progressiva. Embora os desfechos clínicos tardios e as alterações morfológicas estejam bem estabelecidos nessa nefropatia, os mecanismos moleculares que deflagram e aceleram a injúria podocitária ainda não estão completamente elucidados. Podócitos são células altamente especializadas e diferenciadas que revestem a superfície externa dos capilares glomerulares, desempenhando papel essencial na preservação da estrutura e função da barreira de filtração glomerular, sendo alvos frequentes de injúria em muitas nefropatias. A disfunção e depleção dos podócitos glomerulares são, além disso, eventos cruciais implicados na patogênese da progressão da doença renal crônica. Revisaremos a biologia dos podócitos e seu papel na regulação da barreira de filtração glomerular, analisando as principais vias patogênicas envolvidas na lesão podocitária, especialmente relacionadas à nefropatia da DF.

The Role of Marein During Endothelial-Mesenchymal Transition in Diabetic Kidney Disease / Rol de la Mareína Durante la Transición Endotelial-Mesenquimatosa en la Enfermedad Renal Diabética

SUMMARY: Marein is a flavonoid compound that reduces blood glucose and lipids and has a protective effect in diabetes. However, the effect and mechanism(s) of marein on renal endothelial-mesenchymal transition in diabetic kidney disease (DKD) have not been elucidated. In this study, single-cell sequencing data on DKD were analyzed using a bioinformation method, and the data underwent reduced dimension clustering. It was found that endothelial cells could be divided into five subclusters. The developmental sequence of the subclusters was 0, 1, 4, 2, and 3, of which subcluster 3 had the most interstitial phenotype.The expression of mesenchymal marker protein:Vimentin(VIM), Fibronectin(FN1), and fibroblast growth factor receptor 1 (FGFR1) increased with the conversion of subclusters. In db/db mice aged 13-14 weeks, which develop DKD complications after 8-12 weeks of age, marein reduced blood levels of glucose, creatinine, and urea nitrogen, improved structural damage in kidney tissue, and reduced collagen deposition and the expression of FN1 and VIM. Marein also up-regulated autophagy marker:Light chain 3II/I(LC3II/I) and decreased FGFR1 expression in renal tissue. In an endothelial-mesenchymal transition model, a high glucose level induced a phenotypic change in human umbilical vein endothelial cells. Marein decreased endothelial cell migration, improved endothelial cell morphology, and decreased the expression of VIM and FN1. The use of the FGFR1 inhibitor, AZD4547, and autophagy inhibitor, 3-Methyladenine(3-MA), further demonstrated the inhibitory effect of marein on high glucose-induced endothelial-mesenchymal transition by reducing FGFR1 expression and up-regulating the autophagy marker protein, LC3II/I. In conclusion, this study suggests that marein has a protective effect on renal endothelial- mesenchymal transition in DKD, which may be mediated by inducing autophagy and down-regulating FGFR1 expression.

La mareína es un compuesto flavonoide que reduce la glucosa y los lípidos en sangre y tiene un efecto protector en la diabetes. Sin embargo, no se han dilucidado el efecto y los mecanismos de la mareína sobre la transición endotelial- mesenquimatosa renal en la enfermedad renal diabética (ERD). En este estudio, los datos de secuenciación unicelular sobre DKD se analizaron utilizando un método de bioinformación y los datos se sometieron a una agrupación de dimensiones reducidas. Se descubrió que las células endoteliales podían dividirse en cinco subgrupos. La secuencia de desarrollo de los subgrupos fue 0, 1, 4, 2 y 3, de los cuales el subgrupo 3 tenía el fenotipo más intersticial. La expresión de la proteína marcadora mesenquimatosa: vimentina (VIM), fibronectina (FN1) y receptor del factor de crecimiento de fibroblastos. 1 (FGFR1) aumentó con la conversión de subgrupos. En ratones db/db de 13 a 14 semanas de edad, que desarrollan complicaciones de DKD después de las 8 a 12 semanas de edad, la mareína redujo los niveles sanguíneos de glucosa, creatinina y nitrógeno ureico, mejoró el daño estructural en el tejido renal y redujo la deposición y expresión de colágeno de FN1 y VIM. Marein también aumentó el marcador de autofagia: Cadena ligera 3II/I (LC3II/I) y disminuyó la expresión de FGFR1 en el tejido renal. En un modelo de transición endotelial-mesenquimal, un nivel alto de glucosa indujo un cambio fenotípico en las células endoteliales de la vena umbilical humana. Marein disminuyó la migración de células endoteliales, mejoró la morfología de las células endoteliales y disminuyó la expresión de VIM y FN1. El uso del inhibidor de FGFR1, AZD4547, y del inhibidor de la autofagia, 3-metiladenina (3-MA), demostró aún más el efecto inhibidor de la mareína en la transición endotelial-mesenquimal inducida por niveles altos de glucosa al reducir la expresión de FGFR1 y regular positivamente la proteína marcadora de autofagia. , LC3II/I. En conclusión, este estudio sugiere que la mareína tiene un efecto protector sobre la transición endotelial-mesenquimatosa renal en la ERC, que puede estar mediada por la inducción de autofagia y la regulación negativa de la expresión de FGFR1.

Serum autophagy-related gene 5 level in stroke patients: correlation with CD4+ T cells and cognition impairment during a 3-year follow-up

Abstract Autophagy-related gene (ATG) 5 regulates blood lipids, chronic inflammation, CD4+ T-cell differentiation, and neuronal death and is involved in post-stroke cognitive impairment. This study aimed to explore the correlation of serum ATG5 with CD4+ T cells and cognition impairment in stroke patients. Peripheral blood was collected from 180 stroke patients for serum ATG5 and T helper (Th) 1, Th2, Th17, and regulatory T (Treg) cell detection via enzyme-linked immunosorbent assays and flow cytometry. The Mini-Mental State Examination (MMSE) scale was completed at enrollment, year (Y)1, Y2, and Y3 in stroke patients. Serum ATG5 was also measured in 50 healthy controls (HCs). Serum ATG5 was elevated in stroke patients compared to HCs (P<0.001) and was positively correlated to Th2 cells (P=0.022), Th17 cells (P<0.001), and Th17/Treg ratio (P<0.001) in stroke patients but not correlated with Th1 cells, Th1/Th2 ratio, or Treg cells (all P>0.050). Serum ATG5 (P=0.037), Th1 cells (P=0.022), Th17 cells (P=0.002), and Th17/Treg ratio (P=0.018) were elevated in stroke patients with MMSE score-identified cognition impairment vs those without cognition impairment, whereas Th2 cells, Th1/Th2 ratio, and Treg cells were not different between them (all P>0.050). Importantly, serum ATG5 was negatively linked with MMSE score at enrollment (P=0.004), Y1 (P=0.002), Y2 (P=0.014), and Y3 (P=0.001); moreover, it was positively related to 2-year (P=0.024) and 3-year (P=0.012) MMSE score decline in stroke patients. Serum ATG5 was positively correlated with Th2 and Th17 cells and estimated cognitive function decline in stroke patients.

Silencing of matrix metalloprotease-12 delays the progression of castration-resistant prostate cancer by regulating autophagy and lipolysis

Abstract The complex pathogenesis of castration-resistant prostate cancer (CRPC) makes it challenging to identify effective treatment methods. Matrix metalloproteinase (MMP)-12 can degrade elastin as well as various extracellular matrix (ECM) components, which is associated with cancer progression. However, the relationship between MMP-12 and CRPC progression is poorly understood. In this study, we observed the effect of MMP-12 on the progression of CRPC and further explored its potential mechanism of action. High levels of MMP-12 were observed in patients with CRPC. We therefore developed cell co-culture and mouse models to study the function of MMP-12. Silencing MMP-12 in CRPC cells disrupted lipid utilization and autophagy marker expression via the CD36/CPT1 and P62/LC3 pathways, respectively, leading to reduced CRPC cell migration and invasion. Moreover, animal experiments confirmed that MMP-12-knockdown CRPC xenograft tumors exhibited reduced tumor growth, and the mechanisms involved the promotion of cancer cell autophagy and the inhibition of lipid catabolism. According to our results, MMP-12 played important roles in the progression of CRPC by disrupting adipocyte maturation and regulating cancer migration and invasion via the modulation of autophagy and lipid catabolism pathways.

Inhibition of anlotinib-induced autophagy attenuates invasion and migration by regulating epithelial-mesenchymal transition and cytoskeletal rearrangement through ATG5 in human osteosarcoma cells

Abstract The cure rates for osteosarcoma have remained unchanged in the past three decades, especially for patients with pulmonary metastasis. Thus, a new and effective treatment for metastatic osteosarcoma is urgently needed. Anlotinib has been reported to have antitumor effects on advanced osteosarcoma. However, both the effect of anlotinib on autophagy in osteosarcoma and the mechanism of anlotinib-mediated autophagy in pulmonary metastasis are unclear. The effect of anlotinib treatment on the metastasis of osteosarcoma was investigated by transwell assays, wound healing assays, and animal experiments. Related proteins were detected by western blotting after anlotinib treatment, ATG5 silencing, or ATG5 overexpression. Immunofluorescence staining and transmission electron microscopy were used to detect alterations in autophagy and the cytoskeleton. Anlotinib inhibited the migration and invasion of osteosarcoma cells but promoted autophagy and increased ATG5 expression. Furthermore, the decreases in invasion and migration induced by anlotinib treatment were enhanced by ATG5 silencing. In addition, Y-27632 inhibited cytoskeletal rearrangement, which was rescued by ATG5 overexpression. ATG5 overexpression enhanced epithelial-mesenchymal transition (EMT). Mechanistically, anlotinib-induced autophagy promoted migration and invasion by activating EMT and cytoskeletal rearrangement through ATG5 both in vitro and in vivo. Our results demonstrated that anlotinib can induce protective autophagy in osteosarcoma cells and that inhibition of anlotinib-induced autophagy enhanced the inhibitory effects of anlotinib on osteosarcoma metastasis. Thus, the therapeutic effect of anlotinib treatment can be improved by combination treatment with autophagy inhibitors, which provides a new direction for the treatment of metastatic osteosarcoma.

Silencing ANGPT2 alleviates ulcerative colitis by regulating autophagy-mediated NLRP3 inflammasome inactivation via the mTOR signaling pathway

Ulcerative colitis (UC) is a difficult intestinal disease characterized by inflammation, and its mechanism is complex and diverse. Angiopoietin-like protein 2 (ANGPT2) plays an important regulatory role in inflammatory diseases. However, the role of ANGPT2 in UC has not been reported so far. After exploring the expression level of ANGPT2 in serum of UC patients, the reaction mechanism of ANGPT2 was investigated in dextran sodium sulfate (DSS)-induced UC mice. After ANGPT2 expression was suppressed, the clinical symptoms and pathological changes of UC mice were detected. Colonic infiltration, oxidative stress, and colonic mucosal barrier in UC mice were evaluated utilizing immunohistochemistry, immunofluorescence, and related kits. Finally, western blot was applied for the estimation of mTOR signaling pathway and NLRP3 inflammasome-related proteins. ANGPT2 silencing improved clinical symptoms and pathological changes, alleviated colonic inflammatory infiltration and oxidative stress, and maintained the colonic mucosal barrier in DSS-induced UC mice. The regulatory effect of ANGPT2 on UC disease might occur by regulating the mTOR signaling pathway and thus affecting autophagy-mediated NLRP3 inflammasome inactivation. ANGPT2 silencing alleviated UC by regulating autophagy-mediated NLRP3 inflammasome inactivation via the mTOR signaling pathway.

Coenzyme Q10 prevents RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways

Coenzyme Q10 (CoQ10) is a potent antioxidant that is implicated in the inhibition of osteoclastogenesis, but the underlying mechanism has not been determined. We explored the underlying molecular mechanisms involved in this process. RAW264.7 cells received receptor activator of NF-κB ligand (RANKL) and CoQ10, after which the differentiation and viability of osteoclasts were assessed. After the cells were treated with CoQ10 and/or H2O2 and RANKL, the levels of reactive oxygen species (ROS) and proteins involved in the PI3K/AKT/mTOR and MAPK pathways and autophagy were tested. Moreover, after the cells were pretreated with or without inhibitors of the two pathways or with the mitophagy agonist, the levels of autophagy-related proteins and osteoclast markers were measured. CoQ10 significantly decreased the number of TRAP-positive cells and the level of ROS but had no significant impact on cell viability. The relative phosphorylation levels of PI3K, AKT, mTOR, ERK, and p38 were significantly reduced, but the levels of FOXO3/LC3/Beclin1 were significantly augmented. Moreover, the levels of FOXO3/LC3/Beclin1 were significantly increased by the inhibitors and mitophagy agonist, while the levels of osteoclast markers showed the opposite results. Our data showed that CoQ10 prevented RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways in RAW264.7 cells.

The essential role of adenine nucleotide translocase 4 on male reproductive function in mice

Adenine nucleotide translocator 4 (Ant4), an ATP/ADP transporter expressed in the early phases of spermatogenesis, plays a crucial role in male fertility. While Ant4 loss causes early arrest of meiosis and increased apoptosis of spermatogenic cells in male mice, its other potential functions in male fertility remain unexplored. Here, we utilized Ant4 knockout mice to delineate the effects of Ant4-deficiency on male reproduction. Our observations demonstrated that Ant4-deficiency led to infertility and impaired testicular development, which was further investigated by evaluating testicular oxidative stress, autophagy, and inflammation. Specifically, the loss of Ant4 led to an imbalance of oxidation and antioxidants. Significant ultrastructural alterations were identified in the testicular tissues of Ant4-deficient mice, including swelling of mitochondria, loss of cristae, and accumulation of autophagosomes. Our results also showed that autophagic flux and AKT-AMPK-mTOR signaling pathway were affected in Ant4-deficient mice. Moreover, Ant4 loss increased the expression of pro-inflammatory factors. Overall, our findings underscored the importance of Ant4 in regulating oxidative stress, autophagy, and inflammation in testicular tissues. Taken together, these insights provided a nuanced understanding of the significance of Ant4 in testicular development.

Rapamycin attenuates ioversol-induced acute kidney injury in rat models / 基础医学与临床

Objective To investigate the role of autophagy in contrast-induced acute kidney injury(CI-AKI)in rats and to explore its possible mechanism.Methods SD rats were randomly divided into control group,acute kid-ney injury model group(intravenous injection of contrast medium ioversol via tail vein;model),rapamycin(RAPA)group and hydroxychloroquine(HCQ)group.Blood urea nitrogen(BUN)and serum creatinine(Scr)con-tents were measured and the potential change foun in renal pathology was detected by HE staining and microscopy.Transmission electron microscopy was used to observe auto-phagy-related changes in ultrastructure.Western blot was used to observe the expression of microtubule-associated protein 1 light chain 3(LC3)Ⅱ/LC3Ⅰ,ubiquitin-binding protein p62 and Histone deacetylase 4(HDAC4).The expression of HDAC4 was also observed by RT-qPCR.Results Compared with control group,the level of BUN,Scr and HDAC4 expression in the model and HCQ group was increased(P＜0.01),the proximal tubules of the kidney were significantly damaged.In the model group,auto-phagososomes and autolysosomes increased,accompanied by an increase of LC3Ⅱ/LC3Ⅰ and a decrease in the p62 level(P＜0.05,P＜0.01);Compared with model group,there were more autophagosomes and autolysosomes were found in RAPA group(P＜0.01),accompanied by increased LC3Ⅱ/LC3Ⅰratio and decrease in the p62 and HDAC4(P＜0.05,P＜0.01).In contrast,the number of autophagy related structures decreased in HCQ group(P＜0.01),accompanied by the simultaneous increase of LC3Ⅱ/LC3Ⅰ,p62 and the increase of HDAC4(P＜0.01).Conclusions Ioversol may induce autophagy activation,while enhancing autophagy by RAPA alleviates CI-AKI induced renal dysfunction.The mechanism is potentially atributed to the regulation of HDAC4.

Effect of sorafenib induced apoptosis and autophagy on drug resistance in HeLa cells / 基础医学与临床

Objective To explore the effect of sorafenib on HeLa cell proliferation by inducing cell apoptosis and autophagy and its impact on drug resistance.Methods The drug-resistant cell strains were constructed through in-termittent induction method,with concentrations of 0,2.5,5.0,7.5,10.0,15.0,20.0 μmol/L.HeLa cells were incubated with increasing concentrations of sorafenib with each concentration for 1 week.The drug-resistant cell strains with stable passages were collected.MTT assay was used to detect the effect of sorafenib on cell prolifer-ation.Cell cycle distribution was analyzed by flow cytometry.The change in the expression of drug-resistant and ap-optotic genes in the parents and drug-resistant cell strains under different drug concentrations was examined by semi-quantitative PCR.The changes of apoptotic related marker proteins LC3-Ⅰ and LC3-Ⅱ were detected by Westernblot.Results Stable drug-resistant strains were successfully obtained;Drug-treated cells were more blocked in the G1 phase.In drug-resistant cells,the expression of apoptosis suppressor gene Bcl-2 was significantly decreased and the apoptotic gene Bax as well as the drug-resistant genes were all significantly increased(P＜0.05).The LC3-Ⅱ/LC3-Ⅰ ratio of drug-resistant cells was significantly higher than that of parent cells(P＜0.05).Conclusions Sorafenib may block the cell cycle,suppress malignant cell proliferation and promote autophage.On one hand,autophagy participates in the development of cell drug resistance and promotes cell survival.On the other hand,drug-induced autophagy may activate some of apoptotic signaling pathway in drug-resistant cells and promote the reversal of cell drug resistance.

Expression levels and clinical significance of autophagy related proteins in placenta tissues of pregnant women with fetal growth restriction / 基础医学与临床

Objective To explore the expression and clinical significance of autophagy related proteins Beclin-1,microtubule-associated protein 1 light chain 3(LC3)in placenta tissues of pregnant women with fetal growth restric-tion(FGR).Methods A total of 40 pregnant women undergoing delivery due to FGR and 40 pregnant women un-dergoing normal delivery in the Second Affiliated Hospital of Zhengzhou University were enrolled as FGR group and healthy group between August 2022 and August 2023.The general clinical data in the two groups were collected.The levels of Beclin-1 and LC3 mRNA in placenta tissues were detected by PCR,and expressions of Beclin-1 and LC3 proteins were detected by immunohistochemistry.The differences in positive of Beclin-1 and LC3 proteins among patients with different clinical data were analyzed.Results The placental thickness,placental mass and neonatal weight in FGR group were lower than those in healthy group(P＜0.05).The mRNA levels of Beclin-1 and LC3 in FGR group were higher than those in healthy group(P＜0.05)and positive expression rates of Beclin-1 and LC3 proteins were significantly higher than those in healthy group(P＜0.05).There was significant difference in positive expression profile of Beclin-1 and LC3 proteins among patients with different placental thickness,placental mass and neonatal weight(P＜0.05).Conclusions The positive expressions of autophagy genes(Beclin-1 and LC3)are related to FGR,and their specific expression levels are closely related to fetal growth and development.

Lentiviral vector mediated steroidogenic factor-1 silencing regulating decidualization and autophagy of endometrial stromal cells / 解剖学报

Objective To explore the influence and mechanism of lentiviral vector-mediated steroidogenic factor-1(SF-1)silence on decidual process of human endometrial stromal cells(hESCs).Methods The endometrial tissues of patients with endometriosis(EM)and normal pregnant women were collected,and the differential expression of SF-1 was detected by Real-time PCR and immunohistochemical staining.hESCs were isolated from the endometrial tissue of normal pregnancy and identified,hESCs were divided into control group,estradiol(E2)+progesterone(P4)group,short hairpin RAN(shRNA,sh)-normal control(NC)+E2+P4 group,sh-SF-1+E2+P4 group,after the corresponding processing,the morphological changes of hESCs were observed under an inverted microscope.Real-time PCR and Western blotting were used to detect the mRNA expression levels and protein levels of human insulin-like growth factor-binding protein-1(IGFBP-1)and prolactin(PRL)in cells,respectively,flow cytometry was used to determine the cycle distribution of the cells,immunofluorescence staining was used to observe the expression of the intracellular auto-bid microtubule-associated protein light chain 3(LC3),Western blotting was used to determine the protein level of intracellular autophagy-related proteins LC3-Ⅱ,LC3-Ⅰ and Beclin-1.Results The relative expression of SF-1 mRNA and the positive rate of SF-1 protein in endometrial tissue of EM patients were higher than those of normal pregnancy endometrial tissue(P<0.05).Compared with the sh-NC+E2+P4 group,the cells in the sh-SF-1+E2+P4 group were mostly long-spindle,and there was no obvious decidualization.The relative mRNA expression and protein expression of IGFBP1 and PRL were significantly down-regulated(P<0.05),the proportion of cells in G0/G1 phase was significantly decreased,the proportion of cells in S phase was significantly increased(P<0.05),the ratio of LC3-Ⅱ/LC3-Ⅰwas significantly increased,and the relative expression of Beclin-1 protein was also significantly up-regulated(P<0.05).Conclusion The expression of SF-1 in the endometrial tissue of EM patients is increased,and SF-1 silencing mediated by lentiviral vector can inhibit the decidualization process of hESCs,which may be related to regulating the level of autophagy.

Effects of gene silencing Cyclooxygenase-2 cooperated with hyperbaric oxygen on neurological repair and apoptosis, autophagy in cerebral hemorrhage rats / 中华急诊医学杂志

Objective:To investigate the effects of gene silencing inducible cyclooxygenase-2 (COX-2) combined with hyperbaric oxygen (HBO) on neuronal cell edema, apoptosis, autophagy and neural functional recovery in rats with intracerebral hemorrhage.Methods:SPF-grade adult male SD rats ( n=96) were used to establish a cerebral hemorrhage model through stereotactic injection of thrombin VII into the caudate nucleus. They were randomized (random number) into 4 groups ( n=24/group): control group, hyperbaric oxygen (HBO) group, COX-2 RNAi group and combined group (COX-2 RNAi+HBO). The siRNA plasmid targeting silencing COX-2 gene expression was constructed. After group treatment, the four rats were randomly selected from each group for testing in each category. Postoperative day 1, 7, and 14 were assessed using the modified neurological severity score (mNSS) for evaluating neurofunctional deficits. On the 7th day, the water content of the brain tissue was measured using the dry/wet weight method. The blood-brain barrier permeability was assessed using the Evans method. Annexin V and TUNEL assays were employed to assess the apoptotic rate of neural cells. The mRNA expression level of COX-2 in brain tissue was determined using the RT-PCR method. The protein expression levels of Beclin-1, COX-2, aquaporin 4 (AQP-4), B cell lymphoma/lewkmia-2 (Bcl-2), caspase-3, hypoxia-inducible factor-1α (HIF-1α) and matrix metalloprotein-2/9 (MMP-2/9) were detected by Western blot (WB). SPSS software was used for data analysis. One-way ANOVA was used for inter group comparisons and LSD- t test was used for further pairwise comparison. Results:The SD rat intracerebral hemorrhage model and plasmid construction were successfully achieved. The mNSS scores were significantly decreased in COX-2 RNAi, HBO and combined groups compared with control group on the 7th day and 14th day (all P<0.01), especially in combined group ( P<0.01). The contents of Evans blue and the water content of brain tissue of all treatment groups were significantly lower than those in control group (all P<0.05), especially in combined group ( P<0.01). The apoptotic rate of neural cells decreased in all treatment groups compared with the control group (all P<0.05), and the combined group decreased the most ( P<0.01). The mRNA expression levels of COX-2 were significantly decreased in all treatment groups compared with the control group (all P<0.01), and combined group silenced COX-2 expression most obviously ( P<0.05). The results of WB showed that the protein expression levels of Beclin-1, COX-2, AQP-4, Caspase-3, HIF-1α, MMP-2/9 were significantly lower than control group (all P<0.05), while the expression of Bcl-2 was increased in all treatment groups (all P<0.01). Among them, the combined group exhibited the most pronounced trend ( P<0.01). Conclusions:Gene silencing of COX-2 in combination with hyperbaric oxygen therapy can effectively restore neurological function in rats with cerebral hemorrhage. The mechanism may be associated with reduced blood-brain barrier permeability, alleviated brain edema, and inhibition of neuronal apoptosis and autophagy.

Research progress on the mechanism of autophagy's role in perioperative neurocognitive disorders / 临床麻醉学杂志

Perioperative neurocognitive disorders commonly manifest as impaired memory and con-fusion in patients,prolonged hospitalisation,poor prognosis,and increased mortality.Autophagy is closely related to the onset of perioperative neurocognitive disorder.Drugs,aging and inflammation affect autophagy levels by regulating signaling pathways such as mTOR and FOXO1,which are involved in the pathogenesis of perioperative neurocognitive disorder.Autophagy plays an important role in improving postoperative cogni-tive dysfunction and learning memory by modulating neuroinflammation,α-synuclein,and τ protein metabo-lism.This article reviews the main pathways regulating autophagy and the role of autophagy in the occurrence and development of perioperative neurocognitive disorders,in order to seek new targets and ideas for the pathogenesis of perioperative neurocognitive disorders.

Effects of intervention in autophagy regulation of p62-Keap1/Nrf2-GPX4 pathway on ferroptosis and oxaliplatin resistance in colorectal cancer cells / 临床与实验病理学杂志

Purpose To investigate the effect of autophagy intervention on ferroptosis and drug resistance of colorectal canc-er cells and its molecular mechanism.Methods The human colorectal cancer cell lines HCT-8,COLO205,HCT-116,SW620,and SW480 were cultured.HCT-116 cells with moder-ate expression of LC3 were screened,and the expression differ-ences of LC3,p62,Keap1,Nrf2,GPX4 proteins,Fe2+,GSH,and MDA between them and OXA-resistant HCT-116/OXA cell lines were detected.The expression levels of LC3,p62,Keap1,Nrf2,GPX4,Fe2+,GSH and MDA were assessed in HCT-116/OXA cells through the intervention of autophagy and ferroptosis intervention agent combined with oxaliplatin.The proliferative activity and sensitivity to oxaliplatin in each group were detected by CCK-8 assay.Cell growth and invasion ability of each group were detected by plate cloning and Trans well assay.Results LC3,p62 and GPX4 expression levels of HCT-116 cells in the 5 groups were moderate.Compared with HCT-116 cells,HCT-116/OXA was less sensitive to oxaliplatin,and the proteins of p62,Nrf2 and GPX4 were highly expressed,LC3 and Keap1 were lowly expressed,and the expression of Fe2+,GSH and MDA were increased(P＜0.05).The levels of LC3,Keap1 protein,Fe2+and MDA in Rapa and Rapa+Fer-1 groups were higher than those in Fer-1 and control groups,while p62,Nrf2,GPX4 and GSH levels were lower.The expressions of GPX4 pro-tein and GSH in Rapa+Fer-1 group were lower than those in Rapa group(P＜0.05).In the autophagy inhibitor group,LC3,p62,Nrf2,GPX4 and GSH were highly expressed in the CQ and CQ+Erastin groups compared with the control and Eras-tin groups,while Keap1 protein,Fe2+and MDA were low.The levels of GPX4 protein and GSH in Erastin group were lower than those in the other three groups,and the levels of Fe2+and MDA were higher than those in the other three groups(P＜0.05).The combination of autophagy activator OXA showed that Rapa intervention group had higher chemical sensitivity to OXA,less number of migrating cells and lower cell proliferation activity than the other three groups.The sensitivity of Rapa+Fer-1 group to oxaliplatin was lower than that of Rapa group,but higher than that of Fer-1 group and control group(P＜0.05).There was no significant difference between Fer-1 group and con-trol group(P＜0.05).Compared with the control group,the cell activity,migration capacity and clonogenesis capacity of Erastin,CQ+Erastin and CQ groups were decreased when auto-phagy inhibitor was combined with OXA,and the Erastin group was the lowest,while the CQ+Erastin group was higher than the Erastin group,and lower than the CQ group(P＜0.05).Con-clusion In colorectal cancer,autophagy is involved in the regu-lation of ferroptosis,and intervention in autophagy can regulate ferroptosis in colorectal cancer cells through the p62-Keap1/Nrf2-GPX4 pathway,thereby reversing oxaliplatin resistance.

Lycium barbarum polysaccharide inhibits starvation-induced apoptosis of Schwann cells by enhancing autophagy / 神经解剖学杂志

Objective:To investigate the inhibitory effect of lycium barbarum polysaccharide(LBP)on apoptosis of Schwann cells(SCs)and its related mechanisms.Methods:The autophagy model was prepared by starvation treatment of RSC96 cells for 12 h,and the expressions of autophagy related proteins LC3 and p62 were detected by Western Blot.Cell Counting Kit-8(CCK-8)kits were used to detect the optimal concentration of LBP.RSC96 cells were randomly divided into Control group,Starvation group and Starvation+LBP group.The expressions of autophagy associated pro-teins(LC3,p62)and myelin associated proteins(p75NTR,PMP22,S100β)were detected by Western Blot or immu-nofluorescence staining.Annexin V/PI fluorescence staining was used to detect apoptosis of the cells.The cell cycle was analyzed by flow cytometry.Western Blot analysis of phosphorylation levels of pathway proteins Erk1/2 and Akt.Results:CCK8 results showed that the viability of damaged RSC96 cells was the best when LBP was 300 μg/ml.Com-pared with Control group,LC3-Ⅱ/LC3-I levels in Starvation group were significantly increased(P＜0.05).Compared with Starvation group,the proportion of apoptotic and necrotic cells in Starvation+LBP group was significantly de-creased,and the proportion of cells in S and G2/M stages was increased.The expression levels of LC3-Ⅱ,p75NTR,PMP22 and S100β were increased,while the expression levels of autophagy substrate protein p62 were decreased.In-creased expression of pathway protein p-Erk1/2(P＜0.05),while the expression of p-Akt protein decreased slightly.Conclusion:LBP can inhibit the apoptosis of SCs and promote the expression of myelin-related proteins by enhancing autophagy,which is related to the activation of Erk1/2 and/or the inhibition of Akt.

Neurotrophin-3 promotes the recovery of neurological function after cerebral ischemia-reperfusion injury in rats / 神经解剖学杂志

Objective:To explore the role and mechanism of neurotrophin-3(NT-3)in promoting neurological func-tion recovery after cerebral ischemia-reperfusion injury in rats.Methods:Twenty-four SD rats were randomly divided into sham operation group(Sham),middle cerebral artery occlusion/reperfusion(MCAO/R)group,and MCAO/R+NT-3 group.The neurological function scores of rats in each group were evaluated using the modified Garcia score.Western Blot was used to detect the expression of NT-3 and LC3B in brain tissues of rats.Culture dishes with the same density of neurons were randomly divided into normal group(Normal),oxygen-glucose deprivation(OGD)group,OGD+NT-3 group,OGD+NT-3+PF-06273340(TrkC inhibitor)group,OGD+NT-3+ZSTK474(PI3K inhibitor)group,and OGD+NT-3+CCT128930(AKT inhibitor)group.Western Blot was used to detect the expression of TrkC,the phosphorylation of PI3K/AKT,and LC3B in neurons.The morphological changes of neurons and the phenomenon of neuronal autophagy were observed using autophagy-specific fluorescent staining.Results:The animal experiment found that the expression of NT-3 increased in the brain tissue with ischemia-reperfusion injury(P＜0.05),and after the treatment with exogenous NT-3,the modified Garcia score increased(P＜0.05),and the level of autophagy weakened(P＜0.05).The cell experiment found that NT-3 can inhibit neuronal autophagy under ischemic hypoxia and maintain the neuronal morphology to the maximum extent.After using PF-06273340,the expression of p-PI3K and p-AKT de-creased(P＜0.05).After using ZSTK474 and CCT128930,the autophagy-inhibiting effect of NT-3 weakened(P＜0.05).Conclusion:NT-3 inhibits autophagy via the PI3K/AKT signaling pathway to maintain neuronal survival,thereby promoting the recovery of neurological function after cerebral ischemia-reperfusion injury in rats.

Influences of Lupinol on the Proliferation,Apoptosis and Invasion of Cervical Cancer Cells by Regulating PI3K/AKT/mTOR Pathway-Mediated Autophagy / 实用妇产科杂志

Objective:To investigate the influences of lupinol on the proliferation,apoptosis and invasion of cer-vical cancer cells by regulating autophagy mediated by phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway.Methods:The proliferation rate of human cervical cancer cell line HeLa cells treated with 0,10,25,50,70,90 μmol/L lupinol was determined,and the appropriate concentration of lupinol was screened out.HeLa cells cultured in vitro were randomly grouped into control group,low-dose lupinol group,high-dose lupinol group,740 Y-P group(PI3K activator),and high-dose lupinol+740 Y-P group.After group intervention with lupinol and 740 Y-P,MDC fluorescence staining was used to detect the forma-tion of autophagic vacuolation of HeLa cells in each group;western blot was used to detect the expression of au-tophagy and PI3K/AKT/mTOR pathway-related proteins in HeLa cells in each group.HeLa cells cultured in vitro were randomly grouped into control group,low-dose lupinol group,high-dose lupinol group,high-dose lupinol+rapamycin(Rapa),and high-dose lupinol+3-methyladenine(3-MA)group.After the intervention of high dose of lupinol,Rapa and 3-MA,the proliferation of HeLa cells in each group was detected by MTT assay and plate colony formation assay;flow cytometry was used to detect the apoptosis of HeLa cells in each group;transwell assay was used to detect the invasion of HeLa cells in each group;western blot was used to detect the expressions of proliferation,apoptosis and epithelial-mesenchymal transition-related proteins in HeLa cells in each group.Re-sults:Compared with the control group,the relative content of autophagic vacuoles,the protein expressions of Mi-crotubule-associated protein 1A/1 B-light chain 3(LC3)Ⅱ/LC3Ⅰ,and Beclin-1 in the low and high dose lupinol groups were all increased(P＜0.05),the phosphorylated PI3K(p-PI3K)/PI3K,phosphorylated AKT(p-AKT)/AKT,and phosphorylated mTOR(p-mTOR)/mTOR decreased(P＜0.05);the relative content of autophagic vac-uoles,the protein expressions of LC3Ⅱ/LC3Ⅰ,and Beclin-1 in the high-dose lupinol group were further increased compared with the low-dose lupinol group(P＜0.05),the p-PI3K/PI3K,p-AKT/AKT,and p-mTOR/mTOR were further decreased(P＜0.05);the relative content of autophagic vacuoles,the protein expressions of LC3Ⅱ/LC3Ⅰ,and Beclin-1 in 740 Y-P group decreased compared with the control group(P＜0.05),the p-PI3K/PI3K,p-AKT/AKT,and p-mTOR/mTOR increased(P＜0.05).Compared with the high-dose lupinol group,the relative content of autophagic vacuoles,the protein expressions of LC3Ⅱ/LC3Ⅰ,and Beclin-1 in the high-dose lupinol+740 Y-P group decreased(P＜0.05),the p-PI3K/PI3K,p-AKT/AKT,and p-mTOR/mTOR increased(P＜0.05).Com-pared with the control group,the cell proliferation rate,colony formation rate,invasion number,and the protein ex-pressions of proliferating cell nuclear antigen(PCNA),B cell lymphoma 2(Bcl-2)and Vimentin in the low and high dose groups of lupinol were all decreased(P＜0.05),the apoptosis rate,and the protein expressions of Bcl-2 as-sociated x protein(Bax)and zonula occludens protein 1(ZO-1)were all increased(P＜0.05);compared with the low-dose lupinol group,the cell proliferation rate,colony formation rate,invasion number,and the protein expres-sions of PCNA,Bcl-2 and Vimentin in the high-dose lupinol group were further decreased(P＜0.05),the apopto-sis rate,and the protein expressions of Bax and ZO-1 were further increased(P＜0.05).Compared with the high-dose lupinol group,the cell proliferation rate,colony formation rate,invasion number,and the protein expres-sions of PCNA,Bcl-2 and Vimentin in the high-dose lupinol+Rapa group were increased(P＜0.05),the apopto-sis rate,and the protein expressions of Bax and ZO-1 were decreased(P＜0.05);the cell proliferation rate,colo-ny formation rate,invasion number,and the protein expressions of PCNA,Bcl-2 and Vimentin in the high-dose lu-pinol+3-MA group were decreased(P＜0.05),the apoptosis rate,and the protein expressions of Bax and ZO-1 were increased(P＜0.05).Conclusions:Lupinol induces protective autophagy by inhibiting the PI3K/AKT/mTOR pathway,thereby promoting the apoptosis of cervical cancer cells and inhibiting their proliferation and inva-sion.Activation of autophagy attenuates the effects of lupinol on the proliferation,apoptosis and invasion of cervi-cal cancer cells.

Role and potential mechanisms of p62/SQSTM1 on migration and metastasis of non-small cell lung cancer / 实用医学杂志

Objective To investigate the effect and regulatory mechanism of autophagy related multifunc-tional protein p62/SQSTM1 on biological behavior in non-small cell lung cancer(NSCLC).Methods RT-qPCR was used to detect the expression of p62 in normal lung cells and NSCLC cells.CCK-8,wound-healing and Transwell assays were used to detect the effects of inhibition and promotion of p62 expression on the proliferation,migration and invasion in NSCLC cells.Western blotting was used to detect the effects of inhibition and promotion of p62 expression on the expression of apoptosis-related proteins(Bcl-2 and Bax)and autophagy-related proteins(ATG5 and Becline1)in NSCLC cells.A nude mouse transplantation tumor experiment was used to detect the effect of inhibiting p62 expression on the tumor volume and mass of NSCLC cells in vivo.Results Compared with that in normal lung cells,the expression level of p62 in A549 cells was the highest.Cell function experiments in vitro showed that inhibition of p62 expression reduced the abilities of proliferation,migration and invasion in A549 cells,and suppressed autophagy and induced apoptosis.Consistently,p62 overexpression has the opposite effects.In addi-tion,animal experiments in vivo showed that inhibition of p62 expression decreased the tumor volume and mass of tumor-bearing mice.Conclusion p62 could promote the growth of NSCLC A549 cell in vivo and in vitro by modu-lating autophagy.

Changes in serum autophagy markers in infants with retinopathy of prematurity and the clinical value / 实用医学杂志

Objective To study the changes in serum autophagy markers in children with retinopathy of prematurity(ROP)and its clinical value.Methods Premature infants who were screened for ROP in our hospital from January 2020 to December 2022 were selected as the study subjects.Those screened out with ROP at 4-6 weeks of birth were assigned to the ROP group and those without ROP to the control group.The levels of serum autophagy markers LC3-Ⅱ,Beclin-1 and P62 were detected at the 3rd day,1st,2nd and 3rd weeks of birth.The two groups were compared in terms of serum autophagy markers.The diagnostic efficacy of serum autophagy markers on ROP was analyzed.Results There was no significant difference in serum LC3-Ⅱ,Beclin-1 and P62 levels between the ROP group and control group at the 3rd day of birth(P>0.05).At the first,second and third weeks of birth,how-ever,the ROP group showed significantly lower levels of serum LC3-Ⅱ and Beclin-1 but higher level of P62 com-pared to the control group(both P<0.05).The levels of serum LC3-Ⅱ,Beclin-1 and P62 at the first,second and third weeks of birth had diagnostic value for ROP.The children in the ROP group who did not receive mechanical ventilation and oxygen inhalation,and did not develop with sepsis and bronchopulmonary dysplasia showed lower serum LC3-Ⅱ and Beclin-1 levels and higher P62 levels at the first,second and third weeks of birth compared to those without the above-mentioned treatment as well as those complications(all P<0.05).In the ROP group,those with severe ROP showed lower serum LC3-Ⅱ and Beclin-1 levels and higher P62 levels at the 3rd day,and 1st,2nd and 3rd week of birth(all P<0.05).Conclusion The levels of serum autophagy markers in children with ROP show significant changes since the first week of birth,so they have diagnostic efficacy for the diseases.