Expression Of Autophagy-related Proteins Research Articles

MHY1485 promotes adriamycin sensitivity in HepG2 cells by inhibiting autophagy.

MHY1485 is an mTOR activator that inhibits the autophagy process by inhibiting the fusion between autophagosomes and lysosomes. This study aimed to explore the role and mechanism of MHY1485 in hepatocellular carcinoma (HCC) and to provide an in-depth understanding of the mechanisms of autophagy regulation in relation to adriamycin (ADM) resistance, as well as the development of a molecularly targeted autophagy-modulating approach. Here, ADM was used to treat HepG2 cells and construct an ADM-resistant cell model. The HepG2/ADM cell line and HepG2 cells were treated with MHY1485 and ADM, respectively, and the proliferation and apoptosis of HCC cells were detected using CCK8, clone formation, flow cytometry, and 5-ethynyl-2'-deoxyuridine staining assays. Ki-67, mTOR phosphorylation, and LC3A expression were detected by IF staining; the expression or phosphorylation levels of autophagy-related proteins (i.e., GLUT1, PGI, PFK, END, and MTHFD2) and apoptosis-related proteins (caspase-3, caspase-8, and caspase-9) were detected by qPCR and western blotting. The number of autophagosomes was determined by monodansylcadaverine staining. Our results showed that MHY1485 can inhibit the proliferation and growth of liver cancer cells, and that MHY1485 combined with ADM can effectively inhibit the tolerance of HepG2/ADM cells to ADM and enhance the efficacy of ADM. The results of the detection of the autophagy-related protein LC3A also indicated that MHY1485 activates mTOR and can affect the phosphorylation level of ULK1, inhibit autophagy, and enhance the sensitivity of liver cancer cells to adriamycin. In summary, MHY1485 can enhance the sensitivity of adriamycin-resistant cells to adriamycin by activating mTOR and blocking the autophagy process in cells; therefore, mTOR may become a potential target for the treatment of liver cancer.

Quercetin activates autophagy to protect rats ovarian granulosa cells from H2O2-induced aging and injury

Autophagy is closely related to the aging of various organ systems, including ovaries. Quercetin has a variety of biological activities, including potential regulation of autophagy. However, whether quercetin-regulated autophagy activity affects the process of ovarian aging and injury has not been clarified yet. This study explores whether quercetin can resist H2O2-induced aging and injury of granulosa cells by regulating autophagy and its related molecular mechanisms in vitro experiments. The cell viability, endocrine function, cell aging, and apoptosis were detected to evaluate the effects of quercetin and autophagy regulators like 3-methyladenine and rapamycin. The levels of autophagy markers Atg5, Atg12, Atg16L, Lc3B II/I, and Beclin1 were determined by Western blot to assess the effects of quercetin, 3-methyladenine and rapamycin on autophagy. Our results showed quercetin resisted H2O2-induced granulosa cell aging and injury by activating protective autophagy. The treatment of 3-methyladenine and rapamycin confirmed the protective function of autophagy in H2O2-induced granulosa cells. 3-methyladenine treatment inhibited the expression of autophagy markers Atg5, Atg12, Atg16L, Lc3B II/I, and Beclin1 and abolished the positive effects on cell viability, estradiol secretion, and cell apoptosis activated by quercetin. In conclusion, quercetin activates autophagy by upregulating the expression of autophagy-related proteins to resist H2O2-induced aging and injury, which is crucial for stabilizing the function of granulosa cells under oxidative injury conditions and delaying aging. This study may explain the protective effects of quercetin on ovarian aging and injury from the perspective of regulating autophagy.

Placenta-derived mesenchymal stem cells protect against diabetic kidney disease by upregulating autophagy-mediated SIRT1/FOXO1 pathway

Diabetic kidney disease (DKD) is a common chronic microvascular complication of diabetes mellitus. Although studies have indicated the therapeutic potential of mesenchymal stem cells (MSCs) for DKD, the underlying molecular mechanisms remain unclear. Herein, we explored the renoprotective effect of placenta-derived MSCs (P-MSCs) and the potential mechanism of SIRT1/FOXO1 pathway-mediated autophagy in DKD. The urine microalbumin/creatinine ratio was determined using ELISA, and renal pathological changes were detected by special staining techniques. Immunofluorescence was used for detecting the renal tissue expression of podocin and nephrin; immunohistochemistry for the renal expression of autophagy-related proteins (LC3, Beclin-1, SIRT1, and FOXO1); and western blotting and PCR for the expression of podocyte autophagy- and pathway-related indicators. We found that P-MSCs ameliorated renal tubular injury and glomerular mesangial matrix deposition and alleviated podocyte damage in DKD rats. PMSCs enhanced autophagy levels and increased SIRT1 and FOXO1 expression in DKD rat renal tissue, whereas the autophagy inhibitor 3-methyladenine significantly attenuated the renoprotective effect of P-MSCs. P-MSCs improved HG-induced Mouse podocyte clone5(MPC5)injury, increased podocyte autophagy, and upregulated SIRT1 and FOXO1 expression. Moreover, downregulation of SIRT1 expression blocked the P-MSC-mediated enhancement of podocyte autophagy and improvement of podocyte injury. Thus, P-MSCs can significantly improve renal damage and reduce podocyte injury in DKD rats by modulating the SIRT1/FOXO1 pathway and enhancing podocyte autophagy.

Orientin alleviates ox-LDL-induced oxidative stress, inflammation and apoptosis in human vascular endothelial cells by regulating Sestrin 1 (SESN1)-mediated autophagy.

Endothelial cells are a crucial component of the vessel-tissue wall and exert an important role in atherosclerosis (AS). To explore the role of Orientin in AS, human vascular endothelial cells (HUVECs) were induced by oxidized low-density lipoprotein (ox-LDL) to simulate the vascular endothelial injury during AS. Cell viability was detected by CCK-8 assay. Oxidative stress and inflammation related markers were measured using kits, RT-qPCR or western blot. Besides, cell apoptosis was assessed with TUNEL staining and cell autophagy was evaluated by LC3 immunofluorescent staining. Additionally, western blot was utilized to evaluate the expression of Sestrin 1 (SESN1) and proteins in AMPK/mTOR signaling. Afterwards, SESN1 was silenced to determine the expression of autophagy-related proteins. The further application of autophagy inhibitor 3-methyladenine (3-MA) was used to clarify the regulatory mechanism of Orientin on autophagy. Results showed that the decreased viability of HUVECs caused by ox-LDL induction was elevated by Orientin. Oxidative stress and inflammation were also attenuated after Orientin addition in HUVECs under ox-LDL condition. Moreover, Orientin suppressed apoptosis and induced autophagy of HUVECs stimulated by ox-LDL, accompanied by enhanced level of phospho (p)-AMPK and declined level of p-mTOR. Interestingly, SESN1 level was elevated by Orientin, and SESN1 depletion alleviated autophagy and reduced p-AMPK expression but enhanced p-mTOR expression. The further experiments indicated that SESN1 silencing or 3-MA addition reversed the inhibitory effects of Orientin on the oxidative stress, inflammation and apoptosis of HUVECs. Collectively, Orientin could induce autophagy by activating SESN1 expression, thereby regulating AMPK/mTOR signaling in ox-LDL-induced HUVECs.

Ginsenoside Rg3 promotes the survival and migration of trophoblast cells in a rat model of gestational hypertension by regulating mir-100a/insulin growth factor-2 axis

Objective: Preeclampsia (PE) is a common complication during pregnancy. miR-100a is expressed in the placenta and regulates the survival and development of placental cells. Insulin growth factor-2 (IGF-2) may serve as its downstream target. This study investigated the protective mechanisms of ginsenoside Rg3 against PE in rat model. Materials and Methods: LPS-induced rat PE models were suitable for intravenous administration of the highly expressed miR-100a ginsenoside Rg3 lentiviral vector. Human trophoblasts were cultured in vitrofor JEG-3, and PE cell models were constructed. In vivoeffects on tumor growth and apoptosis were observed. Ginsenoside Rg3 was treated with different concentrations of shRNA, miR-100a analogs, inhibitors, or IGF-2. Autophagy and the expression of autophagy-related proteins were examined. Trophoblast activity and migration were determined using Cell Counting Kit-8 and Transwell assays. Both drugs strongly inhibited trophoblasts under normal conditions with some synergy between them. Double-luciferase return assay confirmed the binding affinity of miR-100a for IGF-2. Results: In response to Rg3, autophagy and the expression of autophagy-related proteins LC3-I/II, Beclin1, and SQSTM1 were reduced in PE rat placental trophoblasts. Rg3 inhibited autophagy in JEG-3 cells and promoted JEG-3 survival and migration in a concentration-dependent manner. miR-100a upregulated PE expression. These results suggested that autophagy was a vital signaling system. Rg3 intervention inhibited miR-100a expression and miR-100a downregulated IGF-2 expression in placental tissues and promoted autophagy, thereby inhibiting JEG-3 cell survival and migration. In rats, Rg3 inhibited PE development by regulating the activity of the miR-100a-IGF-2 signaling axis. Conclusion: Ginsenoside Rg3 positively regulates the miR-100a-IGF-2 axis and protects PE rats by inhibiting trophoblastic autophagy and promoting trophoblastic cell survival and migration.

Dasatinib induces apoptosis and autophagy by suppressing the PI3K/Akt/mTOR pathway in bladder cancer cells.

Bladder cancer is a common genitourinary malignant disease worldwide. Dasatinib is a small molecule inhibitor of Src family kinases. We investigated the anticancer effect and putative molecular mechanisms of dasatinib on T24 and cisplatin-resistant T24R2 human bladder cancer cells. Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) and colony formation in dasatinib treated bladder cancer cells. Flow cytometry was used to determined cell cycle arrest and apoptosis. The expression of apoptosis and autophagy related proteins were detected by western blot analysis. In bladder cancer cells, dasatinib significantly reduced cell proliferation, colony formation, and induced G1-phase arrest. Dasatinib triggered apoptosis along with an increased expression of apoptosis-related genes (caspases, PARP, and cytochrome c). Down-regulation of Bcl-2 and up-regulation of Bad, which are hallmarks of apoptosis, were found to play a dominant role in mediating the effects of dasatinib treatment. We further showed that dasatinib inhibits p-Src, p-PI3K, p-Akt, and p-mTOR in bladder cancer cells. Dasatinib also increased the expression of markers of autophagy flux such as LC3-II and p62. These results confirmed that dasatinib is a potent chemotherapeutic drug which induces apoptosis and autophagy by suppressing the PI3K/Akt/mTOR pathway in bladder cancer cells.

Glutamine Modulates Heat Shock Proteins, Endoplasmic Reticulum Stress and Cell Death in Rats with Thioacetamide-Induced Severe Acute Liver Failure

Severe Acute Liver Failure (SALF) is characterized by sudden dysfunction of liver cells in previously healthy persons without known underlying liver disease. Effective therapeutic approaches are necessary in an attempt to minimize the damage resulting from liver failure. Glutamine is considered to be an amino acid that plays many essential metabolic functions. This study was designed to evaluate the action of glutamine in protecting against cellular disturbances resulting from Thioacetamide (TAA)-induced SALF in rats. Two doses of thioacetamide (400 mg/kg) and 3 doses of glutamine (25 mg/kg) (ip) were administered. The experiment lasted 48 hours. Glutamine reduced blood levels of AST and ALT in TAA-treated rats. Histological evaluation indicated that glutamine acted in tissue protection by reducing the inflammatory infiltrate, ballooning and centrilobular necrosis induced by SALF, consequently restoring the hepatic parenchyma. Glutamine increased total protein levels and decreased carbonylated protein levels in the liver of SALF animals. Glutamine also acted by modulating HSF-1 and heat shock proteins expression, as well as by decreasing the expression of proteins involved in endoplasmic reticulum stress (GRP78, ATF6 and CHOP), and modifying the expression of apoptosis-related (Bcl -2, Bax and caspase 3) and autophagy-related (mTOR, Beclin1 and LC3α/β) proteins. These effects related to changes in PI3K, Akt and FOXO3a expression. Data obtained support a potential hepatoprotective role of glutamine in SALF.

Autophagy inhibitor 3-methyladenine attenuates renal injury in streptozotocin-induced diabetic mice.

To investigate whether 3-methyladenine (3-MA) can protect the kidney of streptozotocin (STZ) - induced diabetes mice, and explore its possible mechanism. STZ was used to induce diabetes in C57BL/6J mice. The mice were divided into normal control group (NC), diabetes group (DM), and diabetes+3-MA intervention group (DM+3-MA). Blood glucose, water consumption, and body weight were recorded weekly. At the end of the 6th week of drug treatment, 24-hour urine was collected. Blood and kidneys were collected for PAS staining to evaluate the degree of renal injury. Sirius red staining was used to assess collagen deposition. Blood urea nitrogen (BUN), serum creatinine, and 24-hour urine albumin were used to evaluate renal function. Western blot was used to detect fibrosis-related protein, inflammatory mediators, high mobility group box 1 (HMGB1)/NF-κB signal pathway molecule, vascular endothelial growth factor (VEGF), and podocin, and immunohistochemistry (IHC) was used to detect the expression and localization of autophagy-related protein and fibronectin. Compared with the kidney of normal control mice, the kidney of diabetes control mice was more pale and hypertrophic. Hyperglycemia induces renal autophagy and activates the HMGB1/NF-κB signal pathway, leading to the increase of inflammatory mediators, extracellular matrix (ECM) deposition, and proteinuria in the kidney. In diabetic mice treated with 3-MA, blood glucose decreased, autophagy and HMGB1/NF-κB signaling pathways in the kidneys were inhibited, and proteinuria, renal hypertrophy, inflammation, and fibrosis were improved. 3-MA can attenuate renal injury in STZ-induced diabetic mice through inhibition of autophagy and HMGB1/NF-κB signaling pathway.

Autophagy in Neuroinflammation: A Focus on Epigenetic Regulation.

Neuroinflammation, characterized by the secretion of abundant inflammatory mediators, pro-inflammatory polarization of microglia, and the recruitment of infiltrating myeloid cells to foci of inflammation, drives or exacerbates the pathological processes of central nervous system disorders, especially in neurodegenerative diseases. Autophagy plays an essential role in neuroinflammatory processes, and the underlaying physiological mechanisms are closely correlated with neuroinflammation-related signals. Inhibition of mTOR and activation of AMPK and FOXO1 enhance autophagy and thereby suppress NLRP3 inflammasome activity and apoptosis, leading to the relief of neuroinflammatory response. And autophagy mitigates neuroinflammation mainly manifested by promoting the polarization of microglia from a pro-inflammatory to an anti-inflammatory state, reducing the production of pro-inflammatory mediators, and up-regulating the levels of anti-inflammatory factors. Notably, epigenetic modifications are intimately associated with autophagy and the onset and progression of various brain diseases. Non-coding RNAs, including microRNAs, circular RNAs and long noncoding RNAs, and histone acetylation have been reported to adjust autophagy-related gene and protein expression to alleviate inflammation in neurological diseases. The present review primarily focuses on the role and mechanisms of autophagy in neuroinflammatory responses, as well as epigenetic modifications of autophagy in neuroinflammation to reveal potential therapeutic targets in central nervous system diseases.

P-Cymene and pachymic acid alleviate adalimumab-resistance cell line inflammatory response via inhibiting PI3K/AKT/mTOR signaling pathway

Background: In the management of inflammatory bowel disease, the emergence of drug resistance to monoclonal antibodies has come out as a significant impediment to achieving desired therapeutic outcomes. However, extensive evidence has substantiated that the induction of autophagy holds promise in counteracting the development of drug resistance. In this regard, the utilization of traditional Chinese medicine presents a potential avenue for alleviate drug resistance, specifically through Peilan and Fuling. Consequently, the objective of this study aims to investigate the regulatory effects of p-Cymene and pachymic acid on the autophagy signaling pathway, with the ultimate goal of reducing inflammatory response and alleviating drug resistance. Methods: The RAW 264.7 murine macrophage cell line was subjected to induction using varying concentration gradients of adalimumab in order to establish an adalimumab-resistant cell line. The cell growth curve and viability were assessed using the CCK-8 assay. The expression of cytokines was determined through the implementation of ELISA. The autophagy signaling pathway was evaluated using Western blot analysis. Results: p-Cymene and pachymic acid were found to exert an inhibitory effect on the proliferation of the adalimumab-resistant cell line. Additionally, treatment with p-Cymene and pachymic acid resulted in a decrease in the levels of proinflammatory cytokines, namely TNF-α, IL-1β, and IL-6, while concurrently increasing the level of IL-10. p-Cymene and pachymic acid demonstrated the capacity to suppress the expression of autophagy-related proteins and mRNA including PI3K, AKT, and mTOR. Conclusions: p-Cymene and pachymic acid exhibit the potential to alleviate adalimumab-resistance and ameliorate the inflammatory response, primarily through the activation of the PI3K/AKT/mTOR signaling pathway. These findings suggest that traditional Chinese medicine holds promise as a viable treatment option for mitigating monoclonal antibody resistance in inflammatory bowel disease.

ANRIL regulates retinoblastoma progression via targeting autophagy by miR-328-3p/TSC1/ULK signaling.

Retinoblastoma is the most common primary intraocular malignancy of childhood. The aim of our study was to investigate the role and regulatory mechanism of the long non-coding RNA ANRIL in retinoblastoma. Here, our data demonstrated that ANRIL overexpression inhibited miR-328-3p expression, but promoted expression of autophagy-related proteins (LC3B, ATG5, and BECN1). Then we predicted the binding sites for ANRIL with miR-328-3p, and for miR-328 3p with TSC1/ULK2 3'-UTR, and confirmed the combination of miR-328-3p and ANRIL and TSC1/ULK2 3'-UTR. Importantly, the data showed that ANRIL overexpression promoted TSC1 and ULK2 expression, and inhibited the phosphorylation of mTOR. Finally, our results indicated that ANRIL overexpression facilitated Y79 cell proliferation and cisplatin-induced apoptosis. Our results indicated that ANRIL promoted the proliferation and cisplatin resistance of Y79 cells through activating autophagy by promoting TSC1/ULK2 ex- pression via acting as a miR-328-3p sponge.

Ameliorative effect of bound polyphenols in mung bean coat dietary fiber on DSS-induced ulcerative colitis in mice: the intestinal barrier and intestinal flora.

The consumption of dietary fiber is beneficial for gut health, but the role of bound polyphenols in dietary fiber has lacked systematic study. The aim of this study is to evaluate the ameliorative effect of mung bean coat dietary fiber (MDF) on DSS-induced ulcerative colitis in mice in the presence and absence of bound polyphenols. Compared to polyphenol-removed MDF (PR-MDF), MDF and formulated-MDF (F-MDF,backfilling polyphenols by the amount of extracted from MDF into PR-MDF) alleviated symptoms such as weight loss and colonic injury in mice with colitis, effectively reduced excessive inflammatory responses, and the bound polyphenols restored the integrity of the intestinal barrier by promoting the expression of tight junction proteins. Additionally, bound polyphenols restored the expression of autophagy-related proteins (mTOR, beclin-1, Atg5 and Atg7) and inhibited the excessive expression of apoptotic-related proteins (Bax, caspase-9, and caspase-3). Furthermore, bound polyphenols could ameliorate the dysregulation of the intestinal microbiota by increasing the abundance of beneficial bacteria and inhibiting the abundance of harmful bacteria. Thus, it can be concluded that the presence of bound polyphenols in MDF plays a key role in the alleviation of DSS-induced ulcerative colitis.

Targeting GABARAPL1/HIF-2a axis to induce tumor cell apoptosis in nasopharyngeal carcinoma.

The primary gene mutations associated with nasopharyngeal carcinoma (NPC) are located within the phosphoinositide 3-kinase-mammalian target of rapamycin signaling pathways, which have inhibitory effects on autophagy. Compounds that target autophagy could potentially be used to treat NPC. However, autophagy-related molecular targets in NPC remain to be elucidated. We aimed to examine levels of autophagy-related genes, including autophagy-related 4B cysteine peptidase (ATG4B) and gamma-aminobutyric acid (GABA) type A receptor-associated protein-like 1 (GABARAPL1), in NPC cells and explored their potential role as novel targets for the treatment of NPC. The mRNA and protein expression of autophagy-related genes were detected in several NPC cells. Levels of GABARAPL1 were modified by either overexpression or knockdown, followed by examining downstream targets using RT-qPCR and western blotting. The role of GABARAPL1 in NPC proliferation and apoptosis was examined by flow cytometry. Furthermore, the role of GABARAPL1 was assessed in vivo using a nude mouse xenograft tumor model. The underlying mechanism by which GABARAPL1 regulated nasopharyngeal tumor growth was investigated. Autophagy-related 4B cysteine peptidase (ATG4B), GABARAPL1, and Unc-51-like kinase 1 (ULK1) were significantly down-regulated in multiple NPC cell lines. Overexpression of GABARAPL1 up-regulated the expression of autophagy-related proteins, decreased the level of hypoxia-inducible factor (HIF)-2α, and induced apoptosis in NPC cells. Importantly, overexpression of GABARAPL1 slowed tumor growth. Western blotting showed that autophagy was activated, and HIF-2α was down-regulated in tumor tissues. HIF-2α, as a substrate for autophagic degradation, may play an interesting role during NPC progression.

MiR-125b reverses cisplatin resistance by regulating autophagy via targeting RORA/BNIP3L axis in lung adenocarcinoma.

The platinum-based chemotherapy is one of the most frequently used treatment protocols for lung adenocarcinoma (LUAD), and chemoresistance, however, usually results in treatment failure and limits its application in the clinic. It has been shown that microRNAs (miRNAs) play a significant role in tumor chemoresistance. In this study, miR-125b was identified as a specific cisplatin (DDP)-resistant gene in LUAD, as indicated by the bioinformatics analysis and the real-time quantitative PCR assay. The decreased serum level of miR-125b in LUAD patients was correlated with the poor treatment response rate and short survival time. MiR-125b decreased the A549/DDP proliferation, and the multiple drug resistance- and autophagy-related protein expression levels, which were all reversed by the inhibition of miR-125b. In addition, xenografts of human tumors in nude mice were suppressed by miR-125b, demonstrating that through autophagy regulation, miR-125b could reverse the DDP resistance in LUAD cells, both in vitro and in vivo. Further mechanistic studies indicated that miR-125b directly repressed the expression levels of RORA and its downstream BNIP3L, which in turn inhibited autophagy and reversed chemoresistance. Based on these findings, miR-125b in combination with DDP might be an effective treatment option to overcome DDP resistance in LUAD.

PFOS exposure destroys the integrity of the blood-testis barrier (BTB) through PI3K/AKT/mTOR-mediated autophagy

Perfluorooctanesulfonate or perfluorooctane sulfonic acid (PFOS), a type of perfluorinated compound, is mainly found in consumer products. Exposure to PFOS could cause male reproductive toxicity by causing injury to the blood-testis barrier (BTB). However, the specific mechanisms through which PFOS affects male reproduction remain unclear. The mammalian target of rapamycin (mTOR) is a vital protein kinase that is believed to be a central regulator of autophagy. In this study, we established in vivo and in vitro models to explore the effects of PFOS on the BTB, autophagy, and the regulatory role of the mTOR signaling pathway. Adult mice were developmentally exposed to 0, 0.5, 5, and 10 mg/kg/day PFOS for five weeks. Thereafter, their testicular morphology, sperm counts, serum testosterone, expression of BTB-related proteins, and autophagy-related proteins were evaluated. Additionally, TM4 cells (a mouse Sertoli cell line) were used to delineate the molecular mechanisms that mediate the effects of PFOS on BTB. Our results demonstrated that exposure to PFOS induced BTB injury and autophagy, as evidenced by increased expression of autophagy-related proteins, accumulation of autophagosomes, observed through representative electron micrographs, and decreased activity of the PI3K/AKT/mTOR pathway. Moreover, treatment with chloroquine, an autophagy inhibitor, alleviated the effects of PFOS on the integrity of TM4 cells in the BTB and the PI3K/AKT/mTOR pathway. Overall, this study highlights that exposure to PFOS destroys the integrity of the BTB through PI3K/AKT/mTOR-mediated autophagy.

The neuroprotection of controlled decompression after traumatic epidural intracranial hypertension through suppression of autophagy via PI3K/Akt signaling pathway

Acute intracranial hypertension (AIH) is a common and tricky symptom that inflicts upon patients after traumatic brain injury (TBI). A variety of clinical options have been applied for the management of AIH, such as physiotherapy, medication, surgery and combination therapy. Specifically, controlled decompression (CDC) alleviates the extent of brain injury and reduces the incidence of a series of post-TBI complications, thereby enhancing the prognosis of patients suffering from acute intracranial hypertension. The objective of the present project is to illuminate the potential molecular mechanism that underlies the neuroprotective effects of CDC in a rat model of traumatic epidural intracranial hypertension (TEIH). Herein, we observed the functional recovery, the degree of brain edema, the level of apoptosis, the expressions of neuronal cell autophagy-related signaling pathway proteins (including Akt, p-Akt, LC3 and Beclin-1) in rat TEIH model at 24 h post-surgery. The results showed in comparison with rapid decompression (RDC), CDC reduced the degree of brain edema, diminished the level of cellular apoptosis and enhanced neurological function, and whereas the neuroprotective effect of CDC could be reversed by rapamycin (Rap). The expressions of Beclin-1 and LC3 in CDC group were significantly lower than those of RDC group, and the expression levels of these two proteins were significantly elevated after the addition of Rap. The expression of p-Akt in CDC group was considerably enhanced than RDC group. After the addition of LY294002, a PI3K/Akt pathway inhibitor, p-Akt protein expression was reduced, and the neuroprotective effect of the rats was markedly inhibited. Taken together, our data demonstrate the superior neuroprotective effect of CDC with regard to alleviating early brain edema, improving the neurological status, suppressing apoptosis and inhibiting neuronal autophagy via triggering PI3K/Akt signaling pathway.

Liuwei Dihuang pills attenuate ovariectomy-induced bone loss by alleviating bone marrow mesenchymal stem cell (BMSC) senescence via the Yes-associated protein (YAP)-autophagy axis

Context Liuwei Dihuang pill (LWDH) has been used to treat postmenopausal osteoporosis (PMOP). Objective To explore the effects and mechanisms of action of LWDH in PMOP. Materials and methods Forty-eight female Sprague-Dawley rats were divided into four groups: sham-operated (SHAM), ovariectomized (OVX), LWDH high dose (LWDH-H, 1.6 g/kg/d) and LWDH low dose (LWDH-L, 0.8 g/kg/d); the doses were administered after ovariectomy via gavage for eight weeks. After eight weeks, the bone microarchitecture was evaluated. The effect of LWDH on the differentiation of bone marrow mesenchymal stem cells (BMSCs) was assessed via osteogenesis- and lipogenesis-induced BMSC differentiation. The senescence-related biological indices were also detected using senescence staining, cell cycle analysis, quantitative real-time polymerase chain reaction and western blotting. Finally, the expression levels of autophagy-related proteins and Yes-associated protein (YAP) were evaluated. Results LWDH-L and LWDH-H significantly modified OVX-induced bone loss. LWDH promoted osteogenesis and inhibited adipogenesis in OVX-BMSCs. Additionally, LWDH decreased the positive ratio of senescence OVX-BMSCs and improved cell viability, cell cycle, and the mRNA and protein levels of p53 and p21. LWDH upregulated the expression of autophagy-related proteins, LC3, Beclin1 and YAP, in OVX-BMSCs and downregulated the expression of p62. Discussion and conclusions LWDH improves osteoporosis by delaying the BMSC senescence through the YAP-autophagy axis.

Asperosaponin VI facilitates the regeneration of skeletal muscle injury by suppressing GSK‐3β‐mediated cell apoptosis

AbstractAsperosaponin VI (ASA VI) is a bioactive triterpenoid saponin extracted from Diptychus roots, of Diptyl, and has previously shown protective functions in rheumatoid arthritis and sepsis. This study investigates the effects and molecular mechanisms of ASA VI on skeletal muscle regeneration in a cardiotoxin (CTX)‐induced skeletal muscle injury mouse model. Mice were subjected to CTX‐induced injury in the tibialis anterior and C2C12 myotubes were treated with CTX. Muscle fiber histology was analyzed at 7 and 14 days postinjury. Apoptosis and autophagy‐related protein expression were evaluated t s by Western blot, and muscle regeneration markers were quantified by quantitative polymerase chain reaction. Docking studies, cell viability assessments, and glycogen synthase kinase‐3β (GSK‐3β) activation analyses were performed to elucidate the mechanism. ASA VI was observed to improve muscle interstitial fibrosis, remodeling, and performance in CTX‐treated mice, thereby increased skeletal muscle size, weight, and locomotion. Furthermore, ASA VI modulated the expression of apoptosis and autophagy‐related proteins through GSK‐3β inhibition and activated the transcription of regeneration genes. Our results suggest that ASA VI mitigates skeletal muscle injury by modulating apoptosis and autophagy via GSK‐3β signaling and promotes regeneration, thus presenting a probable therapeutic agent for skeletal muscle injury.

MiR-99b-3p/Mmp13 axis regulates NLRP3 inflammasome-dependent microglial pyroptosis and alleviates neuropathic pain via the promotion of autophagy

BackgroundNeuropathic pain significantly impairs quality of life, and effective interventions are limited. NOD-like receptor thermal protein domain associated protein 3 (NLRP3)-mediated microglial pyroptosis and the subsequent proinflammatory cytokine production are critical in exacerbating pain. Considering microglial pyroptosis as a potential target for developing specific analgesic interventions for neuropathic pain, our study investigated the pathogenesis and therapeutic targets in this condition. MethodsIn vitro experiments involved the co-culture of the immortalized BV-2 microglia cell line with lipopolysaccharide (LPS) to induce microglial pyroptosis. Differentially expressed microRNAs (miRNAs) were identified using high-throughput sequencing analysis. The downstream target genes of these miRNAs were determined through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and the downstream target genes, combined with miRNAs, were predicted and verified through dual luciferase reporter gene assays. In vivo experiments were conducted to construct a chronic constriction injury (CCI) neuropathic pain model in rats and evaluate the analgesic effects of intrathecal injection of an adeno-associated virus vector (AAV) carrying miR-99b-3p. Gene expression was modulated through mimic or siRNA transfection. Western blot analysis assessed the expression of microglial pyroptosis and autophagy-related proteins, whereas RT-qPCR measured changes in proinflammatory cytokines expression. ResultsLPS-stimulated up-regulation of proinflammatory cytokines in microglia, accompanied by NLRP3-dependent pyroptosis, including increased NLRP3, GSDMD-N, Caspase1-p20, and mature-IL-1β expression. High-throughput sequencing analysis revealed 16 upregulated and 10 downregulated miRNAs in LPS-stimulated microglia, with miR-99b-3p being the most downregulated. KEGG analysis revealed that the target genes of these miRNAs are primarily enriched in calcium, FoxO, and mitogen-activated protein kinase (MAPK) signal pathways. Furthermore, overexpression of miR-99b-3p through mimic transfection significantly inhibited the inflammatory response and NLRP3-mediated pyroptosis by promoting autophagy levels in activated microglia. In addition, we predicted that the 3′ untranslated region (UTR) of matrix metalloproteinase-13 (Mmp13) could bind to miR-99b-3p, and knockdown of Mmp13 expression through siRNA transfection similarly ameliorated enhanced proinflammatory cytokines expression and microglial pyroptosis by enhancing autophagy. In vivo, Mmp13 was co-localized with spinal dorsal horn microglia and was suppressed by intrathecal injection of the AAV-miR-99b-3p vector. Moreover, overpressed miR-99b-3p alleviated CCI-induced mechanical allodynia and neuroinflammation while suppressing pyroptosis by enhancing autophagy in the spinal cord of CCI rats. ConclusionmiR-99b-3p exerts analgesic effects on neuropathic pain by targeting Mmp13. These antinociceptive effects are, at least in part, attributed to the promotion of autophagy, thereby inhibiting neuroinflammation and NLRP3-mediated pyroptosis in activated microglia.

Tangzhiqing decoction attenuates cognitive dysfunction of mice with type 2 diabetes by regulating AMPK/mTOR autophagy signaling pathway

Ethnopharmacological relevanceTangzhiqing decoction (TZQD) is an effective prescription developed by Jiangsu Province Hospital of Chinese Medicine for the treatment of diabetes mellitus (DM) and its complications, which has a clear cerebral protective effect on mice with diabetic cognitive dysfunction, but its specific mechanism has not been well elucidated. Aims of the studyThis study aims to verify the protection of TZQD on cognitive function in mice with type 2 diabetes mellitus (T2DM) and explore the possible underlying mechanisms. Materials and methodsSix active ingredients in TZQD were detected using high-performance liquid chromatography analysis. In vivo experiments, the protection of TZQD on cognitive function and hippocampal neurons in type 2 diabetes mice was verified to obtain the optimal intervention dose of TZQD. TZQD and 3-methyladenine (3 MA) respectively or jointly intervened in mice with T2DM for 12 weeks, followed by detecting the cognitive difference, hippocampus cornu ammonis 1 (CA1) region injury, and hippocampal neuronal apoptosis in each group. Simultaneously, the investigation of autophagosome formation and organelle impairment in hippocampal neurons, along with the examination of AMPK/mTOR pathway proteins and autophagy-related proteins, was conducted to elucidate the potential mechanisms, through which TZQD modulates autophagy and enhances cognitive function. In vitro experiments, TZQD-containing serum and AMPK inhibitor Compound C (CC) were used to intervene in mouse hippocampal neuron HT22 cells under high glucose environment, further clarifying the regulatory role of TZQD on the AMPK/mTOR pathway and its impact on HT22 cell apoptosis and autophagy. ResultsIn vivo experiment results showed that TZQD had an obvious hypoglycemic effect. Different doses of TZQD could improve cognitive function and hippocampus damage in diabetes mice, with the middle dose of TZQD showing the best effect. TZQD increased the swimming speed of diabetes mice, improved their spatial recognition and memory ability, and reduced hippocampal neuronal apoptosis, Nissl body injury, and p-tau217 protein deposition. In addition, through transmission electron microscopy (TEM), immunofluorescence, and Western blot (WB) detection, TZQD significantly improved the organelle damage of hippocampal neurons in diabetes mice, promoted the formation of autophagy lysosomes, increased the expression of autophagy-related proteins like Beclin 1, LC3II/LC3I, LAMP1, and LAMP2, reduced the level of P62 and promoted autophagy flow, which, however, were all significantly weakened by 3 MA. Meanwhile, TZQD regulated the expressions of AMPK/mTOR pathway proteins. In vitro experimental study results showed that TZQD can regulate the expression ratio of p-AMPK/AMPK alpha 1 and p-mTOR/mTOR in HT22 cells under high glucose conditions and improved the morphology and vitality of HT22 cells. By employing techniques such as monodansylcadaverine (MDC) staining, Lysosomal red fluorescent probe staining, and Annexin V-FITC/PI double staining, the investigation revealed that TZQD administration resulted in enhanced autophagosome formation, preservation of a lysosomal acidic milieu, and consequent mitigation of HT22 cell apoptosis under high glucose conditions. ConclusionsTZQD can regulate the AMPK/mTOR pathway to activate autophagy to attenuate hippocampal neuronal apoptosis, thereby protecting cognitive function in diabetic mice.