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

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.

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.

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.

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.

Effect of Endoplasmic Reticulum Stress-induced Autophagy on Necrotizing Enterocolitis in Neonatal Rats / 医学研究杂志

Objective To investigate the effect of inhibiting autophagy induced by endoplasmic reticulum stress(ERS)on necrotiz-ing enterocolitis(NEC)in neonatal rats.Methods First,the NEC model of neonatal rats was established.Then,the intestinal epitheli-al cells were isolated and divided into three groups:control group,inhibition group and induction group.The control group was cultured normally,the inhibition group was added with 4-phenylbutyric acid,and the induction group was added with tunicamycin for 24hours.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of the cellular inflammatory cytokines tumor necrosis factor-α(TNF-α)and intestinal fatty acid binding protein(I-FABP)in each group.Real-time quantitative polymerase chain reac-tion(RT-qPCR)was used to detect the mRNA expression level of the markers of ERS glucose regulated protein 78(GRP78)and oxy-gen-regulated protein 150(ORP150).Western blot was used to detect the expression of autophagy related proteins LC3 Ⅱ/Ⅰ and p62.Results Compared with the control group,the expression of p62 in the inhibition group increased significantly,the expression of TNF-α,I-FABP,GRP78,ORP150,LC3 Ⅱ/Ⅰ in the inhibition group was significantly decreased,while the expression of p62 in the induc-tion group was significantly decreased,the expressions of TNF-α,Ⅰ-FABP,GRP78,ORP150,LC3 Ⅱ/Ⅰ were significantly increased,and the differences were statistically significant(P＜0.05).Conclusion Inhibition of ERS induced autophagy activation can alleviate intestinal mucosal injury and inflammatory response in neonatal rats with NEC and improve intestinal barrier function.

Effect of Icariin Regulating mTOR/Akt/CREB Pathway on High Glucose Induced Autophagy and Apoptosis in Podocytes / 医药导报

Objective To investigate the effects of icariin on high glucose-induced autophagy and apoptosis of podocytes,and the regulating effects on mammalian target of rapamycin(mTOR)/serine-threonine kinase(Akt)/cyclic adenosine monophosphate response element binding protein(CREB)pathway.Methods The mouse podocytes MPC5 were taken and divided into five groups:normal control group(5.5 mmol·L-1 glucose),high glucose group(30 mmol·L-1 glucose),icariin group(30 mmol·L-1glucose+5 μmol·L-1icariin),GDC-0349 group(30 mmol·L-1glucose+50 μmol·L-1 GDC-0349),icariin+GDC-0349 group(30 mmol·L-1 glucose+5 μmol·L-1 icariin+50 μmol·L-1 GDC-0349).Cultured for 48 hours,the tetramethylazozolium salt method was used to detect the viability of MPC5 cells;acridine orange staining was used to observe the autophagy of MPC5 cells;apoptosis of MPC5 cells was detected by flow cytometry;Western blotting was used to detect the expression of autophagy[microtubule associated protein one light chain 3(LC3)II,LC3Ⅰ,autophagy-related protein(Beclin-1)],apoptosis[Bcl-2 related X protein(Bax),B cell lymphoma-2(Bcl-2)]and mTOR/Akt/CREB pathway-related proteins of MPC5 cells.Results Compared with the normal control group,the cell viability,expression levels of Bcl-2,phosphorylated mTOR(p-mTOR)/mTOR,phosphorylated Akt(p-Akt)/Akt,phosphorylated CREB(p-CREB)/CREB protein of MPC5 cells in the high glucose group were significantly decreased(P＜0.05),the autophagy ability was enhanced,the autophagosome showed orange fluorescence,and the apoptosis rate,LC3Ⅱ/LC3Ⅰ,Beclin-1,Bax protein expression levels were significantly increased(P＜0.05).Compared with the high glucose group,the cell viability,LC3Ⅱ/LC3Ⅰ,Beclin-1,Bcl-2,p-mTOR/mTOR,p-Akt/Akt,p-CREB/CREB protein expression levels of MPC5 cells in icariin group were significantly increased,the autophagy ability was further enhanced,the number of autophagosomes was increased,the autophagosomes showed brick red fluorescence(P＜0.05),the apoptosis rate and Bax protein expression level were significantly decreased(P＜0.05),and the cell viability,LC3Ⅱ/LC3Ⅰ,Beclin-1,Bcl-2,p-mTOR/mTOR,p-Akt/Akt and p-CREB/CREB proteins expression levels of MPC5 cells in GDC-0349 group were significantly decreased,the autophagy ability was weakened,the number of autophagosomes was reduced,the autophagosomes showed orange fluorescence(P＜0.05),and the apoptosis rate and Bax protein expression level were significantly increased(P＜0.05);icariin+GDC-0349 could reverse the effect of icariin on high glucose induced MPC5 cells(P＜0.05).Conclusion Icariin promotes elevated glucose-induced podocyte autophagy and inhibits apoptosis by activating the mTOR/Akt/CREB pathway.

Effect of Wedelolactone on Oxidative Injury in HUVECs Via the PI3K/Akt/mTOR Signaling Pathway / 医药导报

Objective To study the protective effect of Wedelolactone(WEL)against inflammatory injury in human umbilical vein endothelial cells(HUVECs)and its molecular mechanism by inducing PI3K/Akt/mTOR.Methods The model of atherosclerosis(AS)oxidative stress injury in HUVECs was induced with 200 μmol·L-1 of hydrogen peroxide for 24 h.The experimental groups were as follows:normal control group,DMSO(dimethyl sulfoxide)group,H2O2 group,and WEL group.MTT was used to measure the cell survival rate of each group;flow cytometry was used to assess intracellular ROS levels;fluorescence microscopy was used to detect the expression of p62 protein;immunoblotting assay was used to determine the protein expression levels for apoptosis-related proteins associated with PI3K/Akt/mTOR signaling pathway and autophagy-related proteins.Results Compared with the H2 O2 group,the HUVEC cell survival rate was significantly inhibited in the WEL group(P＜0.05).ROS production was significantly lower,and the protein expressions of SOD1 and p62 were significantly increased in the WEL group as compared to the hydrogen peroxide group.The protein expression of p-mTOR,p-Akt,and p-PI3K was significantly decreased in hydrogen peroxide(P＜0.01);In the WEL experiment,p-mTOR,p-Akt,and p-PI3K were increased significantly in the post-injury HUVECs(P＜0.01).Conclusion Wedelolactone inhibits HUVECs'autophagy by suppressing H2O2-induced inflammatory damage in HUVECs,which may be related to the fact that WEL promotes the phosphorylation of PI3K,Akt,and mTOR proteins,inhibits autophagy and thus resists oxidative stress damage in HUVECs cells.

Melatonin Enhances the Chemosensitivity to Gemcitabine in Pancreatic Cancer(PANC-1)Via the Ferroptosis and Autophagy Pathways / 医药导报

Objective To explore the effect and potential mechanisms of melatonin combined with gemcitabine on the chemosensitivity of human pancreatic cancer cell line PANC-1.Methods Human pancreatic cancer cell line PANC-1 was trea-ted with gemcitabine alone or in combination with melatonin.Cell viability was assessed using CCK-8.Effect of melatonin and gem-citabine alone or in combination on the clonogenic capacity of PANC-1 cells were observed through colony formation experiments.Scratch assays and transwell experiments were conducted to evaluate cell migration ability.Reactive oxygen species(ROS)and mitochondrial membrane point JC-1 assay kit were used to determine reactive oxygen species synthesis and membrane potential levels.Intracellular Fe2+level was measured using ferrous ion fluorescent probe.The protein expression levels of LC3,P62,GPX4 and SLC7A11 in different treatment groups were detected by immunofluorescence and Western blotting.Results CCK-8 results showed that the viability of PANC-1 cells was inhibited by gemcitabine alone after 48 h and 72 h of treatment in a time-and dose-dependent manner.The cell viability of gemcitabine combined with melatonin group was significantly lower than that of gemcitabine group,and the cell viability decreased with the increase of melatonin concentration.Scratch assays,transwell experiments,and plate colony formation assay results demonstrated that the proliferation and migration of cells in the gemcitabine combined with the me-latonin group were significantly inhibited compared with the gemcitabine group.The levels of reactive oxygen species and Fe2+in PANC-1 in gemcitabine combined with the melatonin group were higher than those in the gemcitabine group,and the mitochondri-al membrane potential was significantly decreased(P＜0.01).Western blotting and immunofluorescence results showed that the ra-tio of autophagy-related protein LC3-Ⅱ/LC3-Ⅰ in gemcitabine combined with the melatonin group was lower than that in the gem-citabine group,and the expression of P62 was up-regulated,and the expression of anti-iron death-related protein GPX4 and SLC7A11 was significantly inhibited(P＜0.05),suggesting that melatonin combined with gemcitabine can inhibit autophagy and promote ferroptosis in PANC-1 cells.Conclusion Melatonin enhances the chemosensitivity of pancreatic cancer cell PANC-1 to gemcitabine by inhibiting autophagy and promoting ferroptosis of tumor cells.

Progress in role of m6A modification regulatory enzymes and their binding proteins in cell autophagy / 中国病理生理杂志

Autophagy is an important mechanism to maintain cellular function and metabolism,whereas ab-normal autophagy can cause the advent and worsening of various diseases.N6-Methyladenosine(m6A)RNA methylation is a reversible RNA modification,which is regulated by m6A methyltransferase,m6A demethylase and m6A-binding protein.Studies have shown that autophagy-related genes promote or attenuate autophagy level dependent on the regulation of m6A,and then participate in the process of diseases.This paper reviews the progress of m6A modification regulatory enzymes and their binding proteins in regulating cell autophagy to provide reference for future researches.

Synergistic sensitization of hUMSCs-derived supernatant combined with temozolomide in different glioma cell lines / 中国病理生理杂志

AIM:To explore the synergistic sensitization effect of human umbilical cord mesenchymal stem cell culture supernatant(hUMSC-CM)combined with temozolomide(TMZ)on various glioma cell lines,and to elucidate the underlying mechanisms.METHODS:The hUMSC-CM was harvested using two different serum deprivation tech-niques at 24 and 48 h,and was converted into freeze-dried powder,which was then given to rat malignant glioma cell line RG-2,human astrocytoma cell line U251 and human glioblastoma cell line LN-428 at 5 concentrations(0,1,3,6 and 9 g/L).The effectiveness and sensitivity of hUMSC-CM for inhibiting growth of glioma cells at 24,48 and 72 h were as-sessed using CCK-8 assay.Hematoxylin-eosin(HE)staining combined with CCK-8 assay was employed to evaluate the chemotherapy sensitivity of glioma cells after 48 h of treatment with TMZ at 6 concentrations(0,25,50,100,200 and 400 μmol/L).Two concentrations(3 and 9 g/L)of hUMSC-CM and 3 concentrations(50,100 and 200 μmol/L)of TMZ were chosen for concurrent treatment of glioma cells to assess the proliferation and pathological alterations.TUNEL staining was utilized to detect apoptosis.Flow cytometry was utilized to analyze cell cycle modifications.The expression alterations of apoptosis-inducing proteins,cleaved caspase-3,cleaved caspase-8 and cleaved PARP1,as well as autophagy-inducing proteins beclin-1 and LC3,were examined using Western blot to investigate the synergistic sensitization mechanism of hUMSC-CM combined with TMZ in vitro.RESULTS:The susceptibility of glioma cell lines to hUMSC-CM and TMZ varied,with RG-2 showing the highest sensitivity,followed by U251,and then LN-428.The inhibitory effect of hUMSC-CM(3 and 9 g/L)and TMZ(50,100 and 200 μmol/L)combined treatment on glioma cells was significantly greater than that that of single-agent treatments(P＜0.05),demonstrating a dose-and concentration-dependent enhancement.Notably,the combination of 9 g/L hUMSC-CM(C9)with 50 μmol/L TMZ(T50)effectively suppressed glioma cell growth.CCK-8 as-say indicated a significant reduction of cell viability in C9+T50 group compared with either C9 or T50 alone(P＜0.05).HE staining and TUNEL staining revealed pronounced morphological changes and significant apoptotic features in glioma cells treated with C9+T50.Flow cytometric analysis confirmed that C9+T50 induced cell cycle arrest in glioma cells.Fur-thermore,compared with control group,the levels of cleaved caspase-3,cleaved caspase-8,cleaved PARP1,beclin-1,and LC3-Ⅱ/LC3-Ⅰ were significantly elevated in the C9+T50-treated glioma cells(P＜0.01).CONCLUSION:(1)The concomitant administration of hUMSC-CM and TMZ exerts a broad inhibitory effect on glioma cells,with a synergistic sen-sitization observed across different cell lines.(2)The enhancement of glioma cell sensitivity to TMZ by hUMSC-CM may be attributed to the modulation of caspase-8/caspase-3/PARP1 signaling pathway and the induction of both apoptosis and autophagy in glioma cells.

Role of inhibition of cellular foaming by lipophagy in atherosclerosis / 中国病理生理杂志

Atherosclerosis is a chronic vascular inflammatory disease caused by abnormal lipid metabolism.The formation of lipid-rich foam cells acts as the initial trigger for development of atherosclerotic lesions.Recent studies have shown that lipophagy,a form of selective autophagy,can selectively degrade lipid droplets stored intracellularly and promote cholesterol efflux through the autophagic lysosomal pathway.As a result,intracellular lipid accumulation is re-duced and foaming is inhibited,making lipophagy a potential new target for current anti-atherosclerosis therapy.This arti-cle reviews the crucial role and molecular mechanism of lipophagy in the link between lipid metabolism and atherosclero-sis.Its objective is to outline the regulatory mechanism of lipophagy and present fresh insights for the treatment of athero-sclerotic diseases.

Expression of autophagy-related genes Beclin1 and LC3 in ossified tissues of posterior longitudinal ligament of cervical spine and correlation analysis with osteogenic factors / 中国脊柱脊髓杂志

Objectives:To investigate the expression levels of autophagy-related genes Beclin1 and LC3 in ossified tissues of posterior longitudinal ligament in cervical spine and their correlations with osteogenic factors.Methods:18 posterior longitudinal ligament tissue specimens from cervical ossification of posterior longitudinal ligament(OPLL)patients underwent OPLL surgical removal from October 2020 to May 2021(OPLL group)and 15 tissue specimens of the posterior longitudinal ligament of the cervical spine that were not ossified(non-OPLL group)were collected.The morphological changes of posterior longitudinal ligament tissues were observed by hematoxylin-eosin(HE)staining;the deposition of calcium salts was observed by Von Kossa staining;The mRNA and protein expression levels of Beclin1,microtubule associated protein light chain 3(LC3),and the osteogenic factors runt-related transcription factor 2(RUNX2),bone morphogenetic protein-2(BMP2),and Osterix in the specimens of the two groups were determined with immunohistochemical staining and RT-qPCR respectively;Pearson correlation analysis was used to explore the relationship between Beclin1,LC3,RUNX2,BMP2,and Osterix.Results:Compared with the non-OPLL group,the cell morphology in the OPLL group was larger and irregular in shape,and the nuclei were more obvious;Von Kossa staining showed that there was no obvious calcium salt deposition in the non-OPLL group,whereas brownish-black calcium salts could be seen in the OPLL group,which were aggregated into flakes or clusters;The expression levels of Beclin1,LC3,RUNX2,BMP2 and Osterix proteins and mRNA in the OPLL group were higher than those in the non-OPLL group(P＜0.05);Beclin1 mRNA expression level was significantly correlated with BMP2,RUNX2 and Osterix(P＜0.05,r＞0.5),while LC3 was not correlated with osteogenic factors.Conclusions:The autophagy-related genes Beclin1 and LC3 are significantly overexpressed in ossified tissues of posterior logitudinal ligament of cervical spine,and Beclin1 is closely related to posterior longitudinal ligament osteogenesis.

Influence of resveratrol on apoptosis in knee osteoarthritis rats by regulating autophagy mediated by SIRT1/AMPK signaling pathway / 中国免疫学杂志

Objective:To investigate the effect of resveratrol on apoptosis of chondrocytes in rats with knee osteoarthritis(KOA)through autophagy mediated by silent information regulator 1(SIRT1)/adenylate activated protein kinase(AMPK)signaling pathway.Methods:Fifty healthy Wistar rats were randomly separated into control group,model group,resveratrol group,resveratrol+ SIRT1 inhibitor group,and autophagy activator group,with 10 rats per group.Except for the control group,the other rats were injected with Freund's complete adjuvant to establish the KOA rat model,resveratrol group,resveratrol+AMPK inhibitor group,and autophagy activator group were treated with 10 μmol/kg resveratrol,10 μmol/kg resveratrol+10 mg/kg EX527,2 mg/kg rapamycin,respectively.After 4 weeks,the grade of Lequesne MG knee joint of rats were observed;the levels of IL-6 and tumor necrosis factor-β(TNF-β)in rat knee joint fluid were measured;HE staining and TUNEL staining were used to observe the morphology and apoptosis of rat knee cartilage;transmission electron microscope was used to observe the autophagy in rat chondrocytes;Western blot was performed to determine the protein expressions of SIRT1,p-AMPK,AMPK,LC3 and Beclin-1.Results:Compared with control group,the local reaction,gait reaction,joint activity,and joint swelling of model group were increased;compared with model group,the local response,gait response(P<0.05),joint activity,and joint swelling in resveratrol group and autophagy activator group were reduced(P<0.05).Compared with control group,the cartilage tissue cells in model group were disordered and rough,with fibrotic degeneration,marginal humeral bulge,reduced organelles,and vacuolar degeneration,the number of autophagosomes was increased,the levels of IL-6 and TNF-β in knee joint fluid,chondrocyte apoptosis rate,Beclin-1 and LC3B/A were increased(P<0.05),the SIRT1 and p-AMPK/AMPK in cartilage tissue were decreased(P<0.05);compared with model group,resveratrol group and autophagy activator group showed improvement in the disordered arrangement of cartilage tissue cells and the marginal humeral bulge,the number of autophago-somes was increased,the levels of IL-6 and TNF-β in knee joint fluid,and the apoptosis rate of chondrocytes were decreased(P<0.05),the levels of SIRT1,p-AMPK/AMPK,Beclin-1 and LC3B/A in cartilage tissue were increased(P<0.05);SIRT1 inhibitor could reverse the protective effect of resveratrol group on rat chondrocytes.Conclusion:Resveratrol maybe autophagic KOA rat chon-drocyte apoptosis mediated by activating SIRT1/AMPK pathway,which can be reversed by SIRT1 inhibitor.

Effects of emodin on autophagy in fibroblast-like synoviocytes in rheumatoid arthritis / 中国比较医学杂志

Objective To study the cGAS/STING signaling pathway and investigate the potential effect of emodin(EMD)on autophagy of human rheumatoid arthritis fibroblast synovial cells(MH7A).Methods CCK-8 method was used to detect MH7A cell proliferation,and the experimental concentration of EMD was screened according to cell survival rate.Then,autophagy inhibitor 3-MA was added to further verify the effect of EMD on autophagy.Autophagy of MH7A cells was detected via the monodansylcadaverine staining method.Protein expression levels of cGAS,STING,p-STING,LC3-Ⅰ,LC3-Ⅱ,P62 and Beclin-1 were detected by Western blot.Results Monodansylcadaverine staining indicated that EMD enhanced the autophagy of MH7A cells.Western blot indicated that EMD decreased the expression of autophagy related proteins cGAS,STING,p-STING and P62,and increased that of LC3-Ⅱ and Beclin-1 in MH7A cells.After addition of the autophagy inhibitor 3-MA,the expression of P62 protein in MH7A cells increased,while that of LC3-Ⅱ and Beclin-1 decreased.Conclusions EMD may accelerate autophagy and inhibit MH7A cell proliferation by down-regulating cGAS/STING signaling pathway proteins.

Study on the mechanism of improving islet β-cell function in patients with type 2 diabetes mellitus by Alogliptin benzoate / 中国糖尿病杂志

Objective To investigate the effect of Alogliptin benzoate on the serum autophagy markers in type 2 diabetes mellitus(T2DM)patients.Methods Eighty newly diagnosed T2DM patients who visited the Department of Endocrinology in Baoding No.1 Central Hospital from December 2021 to October 2022 were randomly divided into a group treated with Metformin(Met group,n=40)and a group treated with Met and Alog(Met+Alog group,n=40).The differences in BMI,WHR,FPG,HbA1c,Atg7 and Beclin-1 between two groups before and after 12 weeks of treatment were compared.Results After treatment,the levels of Atg7 and Beclin-1 increased in both groups(P<0.05),while FPG,HbA1c and HOMA-IR decreased(P<0.05).After treatment,Atg7,Beclin-1 and HDL-C in Met+Alog group were higher than those in Met group(P<0.05).Pearson correlation analysis showed that Atg7 was negatively correlated with BMI,FPG and HbA1c(P<0.05);Beclin-1 was positively correlated with HDL-C(P<0.05),and negatively correlated with BMI,FPG,HbA1c,and TG(P<0.05).Meta linear regression analysis showed that BMI was the influencing factor of Atg7,while BMI and HDL-C were the influencing factors of Beclin-1.Conclusion Alogliptin benzoate may improve islet β cell function by up-regulating the expression of autophagy related factors Atg7 and Beclin-1 in patients with T2DM.

Rubescensine A reduces podocyte damage induced by high glucose through the autophagy pathway mediated by AMPK/SIRT1 pathway / 中国糖尿病杂志

Objective To investigate the mechanism that Rubescensine A reduces the podocyte damage induced by high glucose(HG)through the autophagy pathway mediated by AMP activated protein kinase/silent information regulator 1(AMPK/SIRT1)pathway.Methods Human glomerular podocytes were cultured in vitro,and randomly divided into Control group(Con),HG group,hydroxychloroquine(HCQ)group,and Rapamycin(RAP)group.CCK-8 was used to detect cell viability.Western blotting was used to detect cell apoptosis and podocyte injury related protein expression in each group.The podocyte model induced by high glucose(HG)was treated with Rubescensine A(Rub A)at different concentrations and the optimal concentration was selected.Then,human glomerular podocytes were randomly divided into Con group,HG group,Rub A group,Compound C group,and Rub A+Compound C group.The expression of autophagy,AMPK/SIRT1 pathway related proteins were detected in each group.Results Compared with Con group,the podocyte viability and the protein expressions of Synaptopodin and Bcl-2 was significantly reduced(P<0.05),while the protein expressions of Desmin and Bax were significantly increased in HG group(P<0.05).Compared with the HG group,all indicators were relieved in RAP group.However,the levels of all indicators were worsened in HCQ group.Compared with Con group,the expression levels of Desminand Bax proteins in podocytes were significantly increased(P<0.05),and the podocyte viability,number of autophagosomes,the expression levels of Synaptopodin,Bcl-2,microtubule associated protein light chain 3(LC3)II/I,Beclin-1,p-AMPK/AMPK and SIRT1 proteins were significantly reduced in HG group(P<0.05).Compared with HG group and Rub A+Compound C group,the above indicators were improved in Rub A group.Compound C group reversed the protective effect of Rub A.Conclusion Rubescensine A can promote autophagy by activating AMPK/SIRT1 pathway,thereby reduce podocyte damage induced by high glucose.