Autophagy-mediated degradation of integumentary tapetum is critical for embryo pattern formation.

Autophagy modulates the degradation and recycling of intracellular materials and contributes to male gametophyte development and male fertility in plants. However, whether autophagy participates in seed development remains largely unknown. Here, we demonstrate that autophagy is crucial for timely programmed cell death (PCD) in the integumentary tapetum, the counterpart of anther tapetum, influencing embryo pattern formation and seed viability. Inhibition of autophagy resulted in delayed PCD of the integumentary tapetum and defects in embryo patterning. Cell-type-specific restoration of autophagic activities revealed that the integumentary tapetum plays a non-autonomous role in embryo patterning. Furthermore, high-throughput, comprehensive lipidomic analyzes uncovered an unexpected seed-developmental-stage-dependent role of autophagy in seed lipid metabolism: it contributes to triacylglycerol degradation before fertilization and to triacylglycerol biosynthesis after fertilization. This study highlights the critical role of autophagy in regulating timely integumentary tapetum PCD and reveals its significance in seed lipid metabolism and viability.

[Effect of Bushen Huoxue（） recipe on autophagy of ovariectomized rat chondrocytes based on Akt/mTOR signaling pathway].

OBJECTIVE: To investigate whether Bushen Huoxue recipe can protect articular cartilage by regulating Akt/mTOR signaling pathway to promote the autophagy of chondrocytes in ovariectomized rats. METHODS: Among 30 SPF 12-week-old female SD rats weighing （247.0±7.0） g, 6 were randomly selected as the blank control group, and the remaining rats were randomly divided into model group, BSHXR-L group, BSHXR-M group and BSHXR-H group, with 6 rats in each group. The protective effect of Bushen Huoxue recipe on articular cartilage injury in rats was determined by visual observation score, muscovine O-solid green staining and immunohistochemistry. The expression of autophagy related proteins was detected by Western-blot, and the relative expression of Akt, mTOR and downstream autophagy genes was detected by qPCR. RESULTS: After modeling, BSHXR （L, M, H） groups could alleviate the histological damage of cartilage. Immunohistochemistry showed that the expression of Collagen-Ⅱand Aggrecan gradually increased, and the expression of MMP-13 gradually decreased, and the differences between BSHXR-M and BSHXR-H groups and model group were statistically significant （P<0.05）. The results of Western-blot showed that the autophagy pathway proteins p-Akt/Akt and p-mTOR/mTOR were inhibited in the BSHXR（L, M, H） groups, and the expressions of downstream proteins Beclin-1 and LC3Ⅱwere gradually increased, while p62 was gradually decreased, showing a dose effect. QPCR results showed that BSHXR（L, M, H） groups could promote the relative expression of Beclin-1 and LC3ⅡmRNA, and inhibit the relative expression of p62, Akt, mTOR mRNA, and the differences were statistically significant compared with model group （P<0.05）. CONCLUSION: Bushen Huoxue recipe can enhance the cartilage autophagy response by inhibiting the Akt/mTOR signaling pathway, and then protect the cartilage.

Guasha improves knee osteoarthritis by inhibiting chondrocyte apoptosis and regulating expression of autophagy-related genes and proteins in rats. / åˆ®ç—§å¯¹è†å ³èŠ‚éª¨å ³èŠ‚ç‚Žå¤§é¼ è½¯éª¨ç»†èƒžå‡‹äº¡åŠè‡ªå™¬çš„å½±å“.

OBJECTIVES: To observe the effect of Guasha on inflammation factors, apoptosis and autophagy in the cartilage tissue of knee joint in rats with knee osteoarthritis (KOA), so as to explore its mechanisms underlying improvement of KOA. METHODS: A total of 51 male SD rats were randomized into three groups：blank control, KOA model and Guasha (n= 17 in each group) . The rats in the blank control group received intra-articular injection of 0.9% NaCl solution in the right knee joint. The KOA model was established by intraarticular injection of glutamate sodium iodoacetic acid in the right knee joint. For rats of the Guasha group, Guasha (at a frequency of 1 time/s, and an applied pressure of 0.3-0.5 kgf) was applied to "Yanglingquan" (GB34) and "Xuehai"(SP10) areas of the right leg, once every other day, for 7 consecutive sessions. The circumference of the right knee was measured, The histopathological changes of right knee cartilage were observed after H.E. staining. The contents of inflammatory factors interleukin (IL)-1ß and tumor necrosis factor (TNF)-α in the right knee articular cartilage tissue were assayed using ELISA. The expression levels of autophagy-related key molecule Beclin-1 (homologous series of yeast Atg6), light chain protease complication 3 type II/I (LC3II/LC3 I), ubiquitin binding factor 62 (P62) and cysteine aspartate protease-3 (Caspase-3) mRNAs and proteins of the right knee articular cartilage tissue were measured using real-time fluorescent quantitative PCR and Western blot, separately. The apoptosis of chondrocytes was assayed using TUNEL staining, and the immunoactivity of LC3 determined using immunofluorescence staining. RESULTS: After modeling, the right knee circumfe-rence of the model and Guasha groups was significantly increased compared with the blank control group (P<0.01), and after the intervention, the knee circumference of the Guasha group was markedly decreased in comparison with that of the model group (P<0.05). Results of H.E. staining showed obvious degeneration and defects in the cartilage tissue, necrosis of a large number of chondrocytes, fibrous hyperplasia, accompanied by inflammatory cell infiltration, osteoclast increase, fibroplasia and bone trabecular destruction in the model group, which was relatively milder in the Guasha group. Compared with the blank control group, the expression of Beclin-1 and LC3 mRNAs and proteins, and LC immunofluorescence intensity in the right knee articular cartilage tissue were significantly down-regulated (P<0.01, P<0.001), whereas the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, contents of IL-1ß and TNF-α in the right knee articular cartilage tissue considerably increased (P<0.01, P<0.001) in the model group. In contrast to the model group, the Guasha group had an apparent increase in the expression levels of Beclin-1 and LC3 mRNAs and proteins and LC immunofluorescence intensity in the right knee articular cartilage tissue (P<0.05), and a pronounced decrease in the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, and contents of IL-1ß and TNF-α in the right knee articular cartilage tissue (P<0.05, P<0.01). CONCLUSIONS: Guasha stimulation of GB34 and SP10 can improve joint cartilage damage in KOA rats, which may be associated with its functions in inhibiting the excessive release of inflammatory factors and apoptosis, possibly by down-regulating the expression of P62 and Caspase-3 mRNAs and proteins and up-regulating the expression of Beclin-1 and LC3 mRNAs and proteins, and by promoting autophagy of chondrocytes.

Low Selenium and Low Protein Exacerbate Myocardial Damage in Keshan Disease by Affecting the PINK1/Parkin-mediated Mitochondrial Autophagy Pathway.

OBJECTIVE: Keshan disease (KD) is a myocardial mitochondrial disease closely related to insufficient selenium (Se) and protein intake. PTEN induced putative kinase 1 (PINK1)/Parkin mediated mitochondrial autophagy regulates various physiological and pathological processes in the body. This study aimed to elucidate the relationship between PINK1/Parkin-regulated mitochondrial autophagy and KD-related myocardial injury. METHODS: A low Se and low protein animal model was established. One hundred Wistar rats were randomly divided into 5 groups (control group, low Se group, low protein group, low Se + low protein group, and corn from KD area group). The JC-1 method was used to detect the mitochondrial membrane potential (MMP). ELISA was used to detect serum creatine kinase MB (CK-MB), cardiac troponin I (cTnI), and mitochondrial-glutamicoxalacetic transaminase (M-GOT) levels. RT-PCR and Western blot analysis were used to detect the expression of PINK1, Parkin, sequestome 1 (P62), and microtubule-associated proteins1A/1B light chain 3B (MAP1LC3B). RESULTS: The MMP was significantly decreased and the activity of CK-MB, cTnI, and M-GOT significantly increased in each experimental group (low Se group, low protein group, low Se + low protein group and corn from KD area group) compared with the control group (P<0.05 for all). The mRNA and protein expression levels of PINK1, Parkin and MAP1LC3B were profoundly increased, and those of P62 markedly decreased in the experimental groups compared with the control group (P<0.05 for all). CONCLUSION: Low Se and low protein levels exacerbate myocardial damage in KD by affecting the PINK1/Parkin-mediated mitochondrial autophagy pathway.

Electroacupuncture Mediates Fat Metabolism and Autophagy via a Sirt3-Dependent Mechanism in Mice Fed High-Fat Diet.

This study investigates the therapeutic potential of electroacupuncture (EA) on obesity, focusing on its influence on autophagy and energy metabolism, utilizing a high-fat diet (HFD)-induced mouse model. Treatment with EA significantly reduces body weight, fat deposition, and lipid accumulation in HFD-fed mice. Additionally, EA effectively ameliorates metabolic imbalances, reducing blood glucose levels and plasma markers of liver function. At the molecular level, EA enhances the expression of thermogenesis-associated genes in brown adipose tissue and decreases p53 expression, suggesting a decrease in apoptosis. Autophagy in white adipose tissue is inhibited by EA, as demonstrated by the suppression of key autophagy-related proteins. Further experiments highlight the critical role of Sirtuin 3 (Sirt3) in EA's anti-obesity effects. Sirt3 supplementation combined with EA results in reduced body weight, fat deposition, and lipid accumulation, along with modulations in key metabolic indicators. Moreover, EA's modulatory effect on uncoupling protein 1 (Ucp1), Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α), and p53 is found to be Sirt3 dependent. In conclusion, EA exerts beneficial effects against obesity through Sirt3-dependent modulation of autophagy and energy metabolism, indicating a potential therapeutic approach for obesity and related metabolic disorders.

Pingchuanning Decotion Alleviates Bronchial Asthma Airway Inflammation Through ROS/HMGB1/Beclin-1 Mediated Cell Autophagy.

Objective: Bronchial asthma is a prevalent respiratory disorder characterized by airway inflammation. This study aimed to investigate the protective effect of Pingchuanning decoction (PCN) on airway inflammation in bronchial asthma, focusing on the role of autophagy and its underlying molecular mechanism. Methods: Using an in vitro lipopolysaccharide (LPS)-induced inflammatory damage model of human airway epithelial cells (16HBE), we assessed the effect of PCN. Various experiments were performed to evaluate the expression of autophagy-related genes, autophagosome and vesicle counts, and reactive oxygen species (ROS) levels. Results: First, PCN reduced LPS-induced cellular inflammation. Second, PCN decreased the number of autophagosomes and autophagic vesicles. And third, PCN significantly reduced reactive oxygen species (ROS) levels. Most importantly, PCN also down-regulated LPS-induced expression of HMGB1, Beclin-1, and autophagy-related gene 5 (ATG5) while enhancing the expression of B-cell lymphoma 2 (Bcl-2), which further reduced the LC3II/I ratio. Conclusion: PCN reduces the 16HBE inflammatory response by inhibiting the overexpression of ROS/HMGB1/Beclin-1 mediated cell autophagy. Therefore, it may serve as a potential drug for treating bronchial asthma.

Drosophila tweety facilitates autophagy to regulate mitochondrial homeostasis and bioenergetics in Glia.

Mitochondria support the energetic demands of the cells. Autophagic turnover of mitochondria serves as a critical pathway for mitochondrial homeostasis. It is unclear how bioenergetics and autophagy are functionally connected. Here, we identify an endolysosomal membrane protein that facilitates autophagy to regulate ATP production in glia. We determined that Drosophila tweety (tty) is highly expressed in glia and localized to endolysosomes. Diminished fusion between autophagosomes and endolysosomes in tty-deficient glia was rescued by expressing the human Tweety Homolog 1 (TTYH1). Loss of tty in glia attenuated mitochondrial turnover, elevated mitochondrial oxidative stress, and impaired locomotor functions. The cellular and organismal defects were partially reversed by antioxidant treatment. We performed live-cell imaging of genetically encoded metabolite sensors to determine the impact of tty and autophagy deficiencies on glial bioenergetics. We found that tty-deficient glia exhibited reduced mitochondrial pyruvate consumption accompanied by a shift toward glycolysis for ATP production. Likewise, genetic inhibition of autophagy in glia resulted in a similar glycolytic shift in bioenergetics. Furthermore, the survival of mutant flies became more sensitive to starvation, underlining the significance of tty in the crosstalk between autophagy and bioenergetics. Together, our findings uncover the role for tty in mitochondrial homeostasis via facilitating autophagy, which determines bioenergetic balance in glia.

Integrated Multi-Omics Analyses Reveal That Autophagy-Mediated Cellular Metabolism Is Required for the Initiation of Pollen Germination.

Autophagy is an evolutionarily conserved mechanism for degrading and recycling various cellular components, functioning in both normal development and stress conditions. This process is tightly regulated by a set of autophagy-related (ATG) proteins, including ATG2 in the ATG9 cycling system and ATG5 in the ATG12 conjugation system. Our recent research demonstrated that autophagy-mediated compartmental cytoplasmic deletion is essential for pollen germination. However, the precise mechanisms through which autophagy regulates pollen germination, ensuring its fertility, remain largely unknown. Here, we applied multi-omics analyses, including transcriptomic and metabolomic approaches, to investigate the downstream pathways of autophagy in the process of pollen germination. Although ATG2 and ATG5 play similar roles in regulating pollen germination, high-throughput transcriptomic analysis reveals that silencing ATG5 has a greater impact on the transcriptome than silencing ATG2. Cross-comparisons of transcriptome and proteome analysis reveal that gene expression at the mRNA level and protein level is differentially affected by autophagy. Furthermore, high-throughput metabolomics analysis demonstrates that pathways related to amino acid metabolism and aminoacyl-tRNA biosynthesis were affected by both ATG2 and ATG5 silencing. Collectively, our multi-omics analyses reveal the central role of autophagy in cellular metabolism, which is critical for initiating pollen germination and ensuring pollen fertility.

[Cangxi Tongbi Capsules promote chondrocyte autophagy by regulating circRNA＿0008365/miR-1271/p38 MAPK pathway to inhibit development of knee osteoarthritis].

To investigate the mechanism by which Cangxi Tongbi Capsules promote chondrocyte autophagy to inhibit knee osteoarthritis(KOA) progression by regulating the circRNA＿0008365/miR-1271/p38 mitogen-activated protein kinase(MAPK) pathway. The cell and animal models of KOA were established and intervened with Cangxi Tongbi Capsules, si-circRNA＿0008365, si-NC, and Cangxi Tongbi Capsules combined with si-circRNA＿0008365. Flow cytometry and transmission electron microscopy were employed to determine the level of apoptosis and observe autophagosomes, respectively. Western blot was employed to reveal the changes in the protein levels of microtubule-associated protein light chain 3(LC3)Ⅱ/Ⅰ, Beclin-1, selective autophagy junction protein p62/sequestosome 1, collagen Ⅱ, a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS-5), and p38 MAPK. The mRNA levels of circRNA＿0008365, miR-1271, collagen Ⅱ, and ADAMTS-5 were determined by qRT-PCR. Hematoxylin-eosin staining was employed to reveal the pathological changes of the cartilage tissue of the knee, and enzyme-linked immunosorbent assay to measure the levels of interleukin-1ß(IL-1ß) and tumor necrosis factor-alpha(TNF-α). The chondrocytes treated with IL-1ß showed down-regulated expression of circRNA＿0008365, up-regulated expression of miR-1271 and p38 MAPK, lowered autophagy level, increased apoptosis rate, and accelerated catabolism of extracellular matrix. The intervention with Cangxi Tongbi Capsules up-regulated the expression of circRNA＿0008365, down-regulated the expression of miR-1271 and p38 MAPK, increased the autophagy level, decreased the apoptosis rate, and weakened the catabolism of extracellular matrix. However, the effect of Cangxi Tongbi Capsules was suppressed after interfering with circRNA＿0008365. The in vivo experiments showed that Cangxi Tongbi Capsules dose-dependently inhibited the p38 MAPK pathway, enhanced chondrocyte autophagy, and mitigated articular cartilage damage and inflammatory response, thereby inhibiting the progression of KOA in rats. This study indicated that Cangxi Tongbi Capsules promoted chondrocyte autophagy by regulating the circRNA＿0008365/miR-1271/p38 MAPK pathway to inhibit the development of KOA.

Interplay of Impaired Cellular Bioenergetics and Autophagy in PMM2-CDG.

Congenital disorders of glycosylation (CDG) and mitochondrial disorders are multisystem disorders with overlapping symptomatology. Pathogenic variants in the PMM2 gene lead to abnormal N-linked glycosylation. This disruption in glycosylation can induce endoplasmic reticulum stress, contributing to the disease pathology. Although impaired mitochondrial dysfunction has been reported in some CDG, cellular bioenergetics has never been evaluated in detail in PMM2-CDG. This prompted us to evaluate mitochondrial function and autophagy/mitophagy in vitro in PMM2 patient-derived fibroblast lines of differing genotypes from our natural history study. We found secondary mitochondrial dysfunction in PMM2-CDG. This dysfunction was evidenced by decreased mitochondrial maximal and ATP-linked respiration, as well as decreased complex I function of the mitochondrial electron transport chain. Our study also revealed altered autophagy in PMM2-CDG patient-derived fibroblast lines. This was marked by an increased abundance of the autophagosome marker LC3-II. Additionally, changes in the abundance and glycosylation of proteins in the autophagy and mitophagy pathways further indicated dysregulation of these cellular processes. Interestingly, serum sorbitol levels (a biomarker of disease severity) and the CDG severity score showed an inverse correlation with the abundance of the autophagosome marker LC3-II. This suggests that autophagy may act as a modulator of biochemical and clinical markers of disease severity in PMM2-CDG. Overall, our research sheds light on the complex interplay between glycosylation, mitochondrial function, and autophagy/mitophagy in PMM2-CDG. Manipulating mitochondrial dysfunction and alterations in autophagy/mitophagy pathways could offer therapeutic benefits when combined with existing treatments for PMM2-CDG.

CRISPR/Cas9-mediated inactivation of miR-34a and miR-34b/c in HCT116 colorectal cancer cells: comprehensive characterization after exposure to 5-FU reveals EMT and autophagy as key processes regulated by miR-34.

The miR-34a and miR-34b/c encoding genes represent direct targets of the p53 transcription factor, and presumably mediate part of the tumor suppressive effects of p53. Here, we sought to determine their functional relevance by inactivating miR-34a and/or miR-34b/c using a CRISPR/Cas9 approach in the colorectal cancer (CRC) cell line HCT116. Concomitant deletion of miR-34a and miR-34b/c resulted in significantly reduced suppression of proliferation after p53 activation, enhanced migration, invasion and EMT, as well as reduced sensitivity to chemotherapeutics, increased stress-induced autophagic flux, decreased apoptosis and upregulation of autophagy-related genes after 5-FU treatment. However, inactivation of singular miR-34a or miR-34b/c had little effects on the aforementioned processes. RNA-Seq analysis revealed that concomitant deletion of miR-34a/b/c caused EMT signature enrichment, impaired gene repression by the p53-DREAM pathway and elevated autophagy after 5-FU treatment. A gene signature comprised of mRNAs significantly upregulated after combined inactivation of miR-34a and miR-34b/c showed a significant association with the invasive colon cancer subtype CMS4 and poor overall survival in two CRC patient cohorts, and with 5-FU resistance in CRC cell lines. In miR-34a/b/c-deficient cells the upregulated miR-34 target FOXM1 directly induced p62 and ATG9A, which increased autophagy and consequently attenuated apoptosis and rendered the miR-34a/b/c-KO cells more resistant to 5-FU. Inhibition of autophagy by depletion of ATG9A or chloroquine re-sensitized miR-34a/b/c-deficient HCT116 cells to 5-FU. In summary, our findings show a complementary role of miR-34a and miR-34b/c in the regulation of EMT and autophagy which may be relevant for CRC therapy in the future.

The Effects of Cinobufagin on Hepatocellular Carcinoma Cells Enhanced by MRT68921, an Autophagy Inhibitor.

Cinobufagin, a cardiotonic steroid derived from toad venom extracts, exhibits significant anticancer properties by inhibiting Na[Formula: see text]/K[Formula: see text]-ATPase in cancer cells. It is frequently used in clinical settings to treat advanced-stage cancer patients, improving their quality of life and survival time. However, its long-term use can result in multidrug resistance to other chemotherapy drugs, and the exact mechanism underlying this effect remains unknown. Therefore, this study explores the molecular mechanism underlying the anticancer effects of cinobufagin in hepatocellular carcinomas (HCCs), specifically in HepG2 and Huh-7 cells. As determined using transcriptome analysis, cinobufagin-triggered protective autophagy suppressed cell apoptosis in liver cancer HepG2 and Huh-7 cells by inhibiting the phosphoinositide-3-Kinase (PI3K)-AKT serine/threonine kinase (AKT)-mammalian target of rapamycin (mTOR) pathway. Cinobufagin-inhibited cell proliferation, induced apoptosis, and generated cell autophagy by upregulating the expression of MAP1 light chain 3 protein II, Beclin1, and autophagy-related protein 12-5. In addition, the autophagy inhibitor MRT68921 improved the antiproliferative and proapoptotic effects of cinobufagin in the studied cell lines. Overall, this study suggests that combining cinobufagin with an autophagy inhibitor can effectively treat HCC, providing a potential strategy for cancer therapy.

Mechanisms involved in the HMGB1 modulation of tumor multidrug resistance (Review).

Tumor multidrug resistance (MDR) remains one of the most challenging barriers to successful cancer treatment. Several previous studies have suggested that high mobility group box 1 (HMGB1) may be a promising therapeutic target for overcoming cancer drug resistance. Emerging evidence has indicated that HMGB1 functions as a 'double­edged sword' that plays both pro­ and anti­tumor roles in the development and progression of multiple types of cancer. HMGB1 has also been found to be a key regulator of several cell death and signaling pathways, and is involved in MDR by mediating cell autophagy and apoptosis, ferroptosis, pyroptosis and multiple signaling pathways. Additionally, HMGB1 is regulated by a variety of non­coding RNAs (ncRNAs), such as microRNAs, long ncRNAs and circular RNAs that are involved in MDR. Thus far, studies have been conducted to identify strategies with which to overcome HMGB1­mediated MDR by the targeted silencing of HMGB1 and the targeted interference of HMGB1 expression using drugs and ncRNAs. Therefore, HMGB1 is closely associated with tumor MDR and is a promising therapeutic target.

Guizhi Fuling Wan inhibits autophagy of granulosa cells in polycystic ovary syndrome mice via H19/miR-29b-3p.

OBJECTIVE: To investigate the potential molecular mechanism of traditional Chinese medicine Guizhi Fuling Wan (GZFLW) inhibiting granulosa cells (GCs) autophagy in polycystic ovary syndrome (PCOS). METHODS: Control GCs and model GCs were cultured and treated with blank serum or GZFLW-containing serum. The levels of H19 and miR-29b-3p in GCs were detected using qRT-PCR, target genes of miR-29b-3p were identified using luciferase assay. The protein expressions of Phosphatase and tensin homolog (PTEN), Matrix Metalloproteinase (MMP)-2, and Bax were measured using western blot. The level of autophagy was detected via MDC staining, the degree of autophagosomes and autophagic polymers was observed using dual fluorescence-tagged mRFP-eGFP-LC3. RESULTS: GZFLW intervention reduced the expression of autophagy-related proteins PTEN, MMP-2 and Bax, by upregulating the expression of miR-29b-3p and downregulated the expression of H19 (p < .05 or p < .01). The number of autophagosomes and autophagy polymers was significantly decreased by GZFLW treatment. However, the inhibition of miR-29b-3p and overexpression of H19 induced a significant increase in the number of autophagosomes and autophagic polymers, which attenuated the inhibitory effect of GZFLW on autophagy (p < .05 or p < .01). In addition, inhibition of miR-29b-3p or overexpression of H19 can attenuate the effect of GZFLW on the expression of PTEN, MMP-2 and Bax proteins (p < .05 or p < .01). CONCLUSION: Our study found that GZFLW inhibits autophagy in PCOS GCs via H19/miR-29b-3p pathway.

[Acupuncture regulates autophagy in hippocampal neurons of cerebral ischemia/reperfusion injured rats by activating type â ¢ PI3K/Beclin-1 pathway].

OBJECTIVE: To observe the effects of acupuncture on the expression of type Ⅲ phosphatidylinositol 3-hydroxykinase (PI3K) and Beclin-1 in hippocampus of rats with cerebral ischemia/reperfusion injury (CI/RI), so as to explore the mechanism of acupuncture in regulating type Ⅲ PI3K pathway to activate autophagy in the hippocampal neurons of CI/RI rats. METHODS: SD rats were randomly divided into sham operation group (n=11) and operation group. Then after successful modeling, rats in the operation group were randomly divided into model, acupuncture, model+3-MA and acupuncture+3-MA groups, with 11 rats in each group. The model of CI/RI was established by occlusion of the middle cerebral artery. Rats in the model+3-MA and acupuncture+3-MA groups were injected with 3-MA (400 nmol/ 5 µL) 5 µL into the lateral ventricle 30 min before reperfusion. Rats in the acupuncture and acupuncture+3-MA groups were punctured with filiform needles at "Dazhui" (GV14), "Shuigou" (GV26) and "Baihui" (GV20) and stimulated manually once every 15 min. The acupuncture intervention was conducted for 30 min each time, once every 12 h for a total of 7 times. The degree of neurological impairment was evaluated 2 h after reperfusion and after intervention by Garcia score. After intervention, the percentage of cerebral ischemic area was observed by TTC staining, the protein expression levels of type Ⅲ PI3K, Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B), lysosome associated membrane protein 2 (Lamp2) and P62 in ischemic hippocampal tissue were detected by Western blot, the ultrastructure of neurons in ischemic hippocampus was observed by transmission electron microscopy (TEM). RESULTS: Compared with the sham operation group, the Garcia score was decreased (P<0.01), the percentage of cerebral ischemic area was increased (P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, LC3B-Ⅱ/Ⅰ, Lamp2 proteins were decreased (P<0.01), and the expression level of P62 protein was increased (P<0.01) in ischemic hippocampal tissue in the model group. Compared with the model group, the Garcia score was increased (P<0.01), the percentage of cerebral ischemic area was decreased (P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, LC3B-Ⅱ/Ⅰ, Lamp2 proteins were increased (P<0.01, P<0.05) and the expression level of P62 was decreased (P<0.01) in ischemic hippocampal tissue in the acupuncture group； the percentage of cerebral ischemic area was increased (P<0.05), the expressions of type Ⅲ PI3K and Beclin-1 were decreased (P<0.01) and the expression level of P62 protein was increased (P<0.05) in ischemic hippocampal tissue in the mo-del+3-MA group. Compared with the model +3-MA group, the Garcia score was increased (P<0.05), the percentage of cerebral ischemic area was decreased (P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, LC3B-Ⅱ/Ⅰ in ischemic hippo-campal tissue were increased (P<0.01, P<0.05) in the acupuncture+3-MA group. Compared with the acupuncture group, the Garcia score was decreased, the percentage of cerebral ischemic area was increased (P<0.05, P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, Lamp2 proteins were decreased (P<0.01, P<0.05) and P62 protein was increased (P<0.05) in ischemic hippocampal tissue in the acupuncture+3-MA group. The results of TEM showed that the edema of neurons was heavier, and few hypolysosomes existed in the model group; there was no obvious damage to neuronal structure, intracellular matrix was abundant, and a few lysosomes existed in the acupuncture group; the neuronal cells had mild edema and primary lysosomes were present in the acupuncture +3-MA group. CONCLUSION: Acupuncture can improve the symptoms of neurological impairment and reduce the percentage of cerebral ischemic area in rats with CI/RI. The mechanism may be related to regulating type Ⅲ PI3K/Beclin-1 pathway, up-regulating the expressions of autophagy related factors LC3B-Ⅱ and Lamp2, and down-regulating the expression of P62.

YAP1 synergize with YY1 transcriptional co-repress DUSP1 to induce osimertinib resistant by activating the EGFR/MAPK pathway and abrogating autophagy in non-small cell lung cancer.

YAP1 is a well-known core effector of the Hippo pathway in tumors, but its potential role in osimertinib resistance remained unexplored. Our study provides evidence that YAP1 acts as a potent promoter of osimertinib resistance. By inhibiting YAP1 with a novel inhibitor, CA3, and combining it with osimertinib, we observed a significant suppression of cell proliferation and metastasis, induction of apoptosis and autophagy, and a delay in the emergence of osimertinib resistance. Interestingly, CA3 combined with osimertinib executed its anti-metastasis and pro-tumor apoptosis in part through autophagy. Mechanistically, we found that YAP1, in collaboration with YY1, transcriptionally represses DUSP1, leading to the dephosphorylation of the EGFR/MEK/ERK pathway and YAP1 phosphorylation in osimertinib-resistant cells. Our results also validate that CA3, in combination with osimertinib, executes its anti-metastasis and pro-tumor apoptosis partly through autophagy and the YAP1/DUSP1/EGFR/MEK/ERK regulatory feedback loop in osimertinib-resistant cells. Remarkably, our findings illustrate that YAP1 protein is upregulated in patients after osimertinib treatment and osimertinib resistance. Overall, our study confirms that the YAP1 inhibitor CA3 increases DUSP1 with concomitant activation of the EGFR/MAPK pathway and induces autophagy to enhance the efficacy of third-generation EGFR-TKI treatments for NSCLC patients.

Naringin Alleviates Glucose-Induced Aging by Reducing Fat Accumulation and Promoting Autophagy in Caenorhabditis elegans.

Naringin (Nar) is a dihydroflavonoid compound, widely found in citrus fruit and used in Chinese herbal medicine. As a phytochemical, it acts as a dietary supplement that can delay aging and prevent aging-related disease, such as obesity and diabetes. However, its exact mechanism remains unclear. In this study, the high-glucose-induced (HGI) Caenorhabditis elegans model was used to evaluate the anti-aging and anti-obesity effects of Nar. The mean lifespan and fast movement span of HGI worms were extended roughly 24% and 11%, respectively, by Nar treatment. Oil red O staining revealed a significant reduction in fat accumulation and dFP::LGG-labeled worms showed the promotion of autophagy. Additionally, whole transcriptome sequencing and gene set variation analysis suggested that Nar upregulated the lipid biosynthesis and metabolism pathways, as well as the TGF-ß, Wnt and longevity signaling pathways. Protein-protein interaction (PPI) network analysis identified hub genes in these pathways for further analysis. Mutant worms and RNA interference were used to study mechanisms; the suppression of hlh-30, lgg-1, unc-51, pha-4, skn-1 and yap-1 disabled the fat-lowering, lifespan-prolonging, and health-promoting properties of Nar. Collectively, our findings indicate that Nar plays an important role in alleviating HGI-aging and anti-obesity effects by reducing fat accumulation and promoting autophagy.

Xanthine dehydrogenase rewires metabolism and the survival of nutrient deprived lung adenocarcinoma cells by facilitating UPR and autophagic degradation.

Xanthine dehydrogenase (XDH) is the rate-limiting enzyme in purine catabolism by converting hypoxanthine to xanthine and xanthine to uric acid. The altered expression and activity of XDH are associated with the development and prognosis of multiple types of cancer, while its role in lung adenocarcinoma (LUAD) remains unknown. Herein, we demonstrated that XDH was highly expressed in LUAD and was significantly correlated with poor prognosis. Though inhibition of XDH displayed moderate effect on the viability of LUAD cells cultured in the complete medium, it significantly attenuated the survival of starved cells. Similar results were obtained in XDH-knockout cells. Nucleosides supplementation rescued the survival of starved LUAD cells upon XDH inhibition, while inhibition of purine nucleoside phosphorylase abrogated the process, indicating that nucleoside degradation is required for the XDH-mediated survival of LUAD cells. Accordingly, metabolic flux revealed that ribose derived from nucleoside fueled key carbon metabolic pathways to sustain the survival of starved LUAD cells. Mechanistically, down-regulation of XDH suppressed unfolded protein response (UPR) and autophagic flux in starved LUAD cells. Inhibition of XDH decreased the level of amino acids produced by autophagic degradation, which was accompanied with down-regulation of mTORC1 signaling. Supplementation of amino acids including glutamine or glutamate rescued the survival of starved LUAD cells upon knockout or inhibition of XDH. Finally, XDH inhibitors potentiated the anti-cancer activity of 2-deoxy-D-glucose that induced UPR and/or autophagy in vitro and in vivo. In summary, XDH plays a crucial role in the survival of starved LUAD cells and targeting XDH may improve the efficacy of drugs that induce UPR and autophagy in the therapy of LUAD.

Ginsenoside Rg1 attenuates the NASH phenotype by regulating the miR-375-3p/ATG2B/PTEN-AKT axis to mediate autophagy and pyroptosis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is one of the most frequent liver diseases at present, and there is no radical treatment. The consequences of a variety of ginsenoside compounds on this situation have before been reported, however, the specific effect on the monomeric ginsenoside Rg1 (Rg1) and its associated underlying molecular mechanism stay unknown. MATERIAL AND METHODS: In vitro, the cell models were constructed by exposing free fatty acids (FFAs) to HepG2 cells. A methionine and choline deficiency (MCD)-induced NASH mouse model was also established over 5-6 weeks of treatment. Rg1 is a traditional Chinese medicine monomer. These NASH models were treated with Rg1 and analyzed by qRT-PCR, Western Blot, sequencing, Oil red O staining, immunofluorescence, enzyme activity, HE staining, ELISA, double luciferase reporter assay, and immunohistochemistry. RESULTS: Overexpression of ATG2B, an autophagy-related protein, attenuated lipid droplet accumulation and reduces ALT, AST, inflammatory cytokines, hydrogen peroxide, and pyroptosis in established mouse and cellular models of NASH and increased levels of ATP and autophagy. The binding sites of miR-375-3p and ATG2B were verified by bioinformatic prediction and a dual-luciferase reporter gene. Knockdown of miR-375-3p promoted autophagy and inhibited pyroptosis. ATG2B knockdown substantially attenuated the impact of miR-375-3p on NASH. Rg1 appears to regulate the occurrence and development of NASH inflammation through miR-375-3p and ATG2B in vitro and in vivo, and is regulated by PTEN-AKT pathway. CONCLUSIONS: This study showed that Rg1 participates in autophagy and pyroptosis through the miR-375-3p/ATG2B/PTEN-AKT pathway, thereby alleviating the occurrence and development of NASH, for that reason revealing Rg1 as a candidate drug for NASH.

Coupling of autophagy and the mitochondrial intrinsic apoptosis pathway modulates proteostasis and ageing in Caenorhabditis elegans.

Mitochondria preserve metabolic homeostasis and integrate stress signals, to trigger cytoprotective, or cell death pathways. Mitochondrial homeostasis and function decline with age. The mechanisms underlying the deterioration of mitochondrial homeostasis during ageing, or in age-associated pathologies, remain unclear. Here, we show that CISD-1, a mitochondrial iron-sulfur cluster binding protein, implicated in the pathogenesis of Wolfram neurodegenerative syndrome type 2, modulates longevity in the nematode Caenorhabditis elegans by engaging autophagy and the mitochondrial intrinsic apoptosis pathway. The anti-apoptotic protein CED-9 is the downstream effector that mediates CISD-1-dependent effects on proteostasis, neuronal integrity and lifespan. Moreover, intracellular iron abundance is critical for CISD-1 function, since mild iron supplementation is sufficient to decelerate ageing and partly ameliorate the disturbed mitochondrial bioenergetics and proteostasis of CISD-1 deficient animals. Our findings reveal that CISD-1 serves as a mechanistic link between autophagy and the apoptotic pathway in mitochondria to differentially modulate organismal proteostasis and ageing, and suggest novel approaches which could facilitate the treatment of Wolfram Syndrome or related diseases.