Early secreted antigenic target of 6 kDa protein (ESAT-6) is the major virulence factor of Mycobacterium tuberculosis (MTB), which can resist the clearance of MTB in bodies by inhibiting macrophage phagocytosis and autophagy reaction, thus impeding the immune defense function of the body against MTB infection. In addition, ESAT-6-induced apoptosis of macrophage and massive necrosis of innate immune cells can foster MTB proliferation and colonization, leading to systemic MTB infection. Moreover, ESAT-6 hampers the protective immune response of Th1 cells, reducing the secretion of pro-inflammatory cytokines and contributing to immune dysfunction, thus accelerating the course of MTB infection. During the process, the high immunogenicity of ESAT-6 can be leveraged as a dominant antigen in the development of new TB vaccines, making it a promising candidate with broad prospects for further development.
Humans ; Mycobacterium tuberculosis ; Vaccines ; Cytokines ; Apoptosis ; Autophagy ; Sepsis

OBJECTIVE: To explore the mechanism of electroacupuncture (EA) in promoting recovery of the facial function with the involvement of autophagy, glial cell line-derived neurotrophic factor (GDNF), and phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. METHODS: Seventy-two male Sprague-Dawley rats were randomly allocated into the control, sham-operated, facial nerve injury (FNI), EA, EA+3-methyladenine (3-MA), and EA+GDNF antagonist groups using a random number table, with 12 rats in each group. An FNI rat model was established with facial nerve crushing method. EA intervention was conducted at Dicang (ST 4), Jiache (ST 6), Yifeng (SJ 17), and Hegu (LI 4) acupoints for 2 weeks. The Simone's 10-Point Scale was utilized to monitor the recovery of facial function. The histopathological evaluation of facial nerves was performed using hematoxylin-eosin (HE) staining. The levels of Beclin-1, light chain 3 (LC3), and P62 were detected by immunohistochemistry (IHC), immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Additionally, IHC was also used to detect the levels of GDNF, Rai, PI3K, and mTOR. RESULTS: The facial functional scores were significantly increased in the EA group than the FNI group (P<0.05 or P<0.01). HE staining showed nerve axons and myelin sheaths, which were destroyed immediately after the injury, were recovered with EA treatment. The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats (P<0.01); however, EA treatment reversed these abnormal changes (P<0.01). Meanwhile, EA stimulation significantly increased the levels of GDNF, Rai, PI3K, and mTOR (P<0.01). After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist, the repair effect of EA on facial function was attenuated (P<0.05 or P<0.01). CONCLUSIONS EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI. EA may exert this neuroreparative effect through mediating the release of GDNF, activating the PI3K/mTOR signaling pathway, and further regulating the autophagy of facial nerves.
Rats ; Male ; Animals ; Rats, Sprague-Dawley ; Electroacupuncture ; Phosphatidylinositol 3-Kinase/metabolism* ; Facial Nerve Injuries/therapy* ; Phosphatidylinositol 3-Kinases/metabolism* ; Beclin-1 ; Glial Cell Line-Derived Neurotrophic Factor ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Autophagy ; Mammals/metabolism*

Acute hypobaric hypoxic brain damage is a potentially fatal high-altitude sickness. Autophagy plays a critical role in ischemic brain injury, but its role in hypobaric hypoxia (HH) remains unknown. Here we used an HH chamber to demonstrate that acute HH exposure impairs autophagic activity in both the early and late stages of the mouse brain, and is partially responsible for HH-induced oxidative stress, neuronal loss, and brain damage. The autophagic agonist rapamycin only promotes the initiation of autophagy. By proteome analysis, a screen showed that protein dynamin2 (DNM2) potentially regulates autophagic flux. Overexpression of DNM2 significantly increased the formation of autolysosomes, thus maintaining autophagic flux in combination with rapamycin. Furthermore, the enhancement of autophagic activity attenuated oxidative stress and neurological deficits after HH exposure. These results contribute to evidence supporting the conclusion that DNM2-mediated autophagic flux represents a new therapeutic target in HH-induced brain damage.
Mice ; Animals ; Hypoxia ; Oxidative Stress ; Autophagy ; Cognition ; Sirolimus/therapeutic use*

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-1β(IL-1β) and tumor necrosis factor-alpha(TNF-α). The chondrocytes treated with IL-1β 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.
Rats ; Animals ; Chondrocytes ; Osteoarthritis, Knee/pathology* ; RNA, Circular/pharmacology* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; MicroRNAs/metabolism* ; Apoptosis ; Autophagy/genetics* ; Collagen/metabolism*

This study investigated the effect and mechanism of morin in inducing autophagy and apoptosis in hepatocellular carcinoma cells through the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription protein 3(STAT3) pathway. Human hepatocellular carcinoma SK-HEP-1 cells were stimulated with different concentrations of morin(0, 50, 100, 125, 200, and 250 μmol·L~(-1)). The effect of morin on the viability of SK-HEP-1 cells was detected by Cell Counting Kit-8(CCK-8). The effect of morin on the proliferation and apoptosis of SK-HEP-1 cells was investigated using colony formation assay, flow cytometry, and BeyoClick~(TM) EdU-488 with different concentrations of morin(0, 125, and 250 μmol·L~(-1)). The changes in the autophagy level of cells treated with morin were examined by transmission electron microscopy and autophagy inhibitors. The impact of morin on the expression levels of proteins related to the Akt/mTOR/STAT3 pathway was verified by Western blot. Compared with the control group, the morin groups showed decreased viability of SK-HEP-1 cells in a time-and concentration-dependent manner, increased number of apoptotic cells, up-regulated expression level of apoptosis marker PARP, up-regulated phosphorylation level of apoptosis-regulating protein H2AX, decreased number of positive cells and the colony formation rate, an upward trend of expression levels of autophagy-related proteins LC3-Ⅱ, Atg5, and Atg7, and decreased phosphorylation levels of Akt, mTOR, and STAT3. These results suggest that morin can promote apoptosis, inhibit proliferation, and induce autophagy in hepatocellular carcinoma cells, and its mechanism of action may be related to the Akt/mTOR/STAT3 pathway.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis ; Autophagy ; Cell Proliferation ; Cell Line, Tumor ; STAT3 Transcription Factor/metabolism*

This study explored the protective effect of astragaloside Ⅳ(AS-Ⅳ) on oxygen-glucose deprivation(OGD)-induced autophagic injury in PC12 cells and its underlying mechanism. An OGD-induced autophagic injury model in vitro was established in PC12 cells. The cells were divided into a normal group, an OGD group, low-, medium-, and high-dose AS-Ⅳ groups, and a positive drug dexmedetomidine(DEX) group. Cell viability was measured using the MTT assay. Transmission electron microscopy was used to observe autophagosomes and autolysosomes, and the MDC staining method was used to assess the fluorescence intensity of autophagosomes. Western blot was conducted to determine the relative expression levels of functional proteins LC3-Ⅱ/LC3-Ⅰ, Beclin1, p-Akt/Akt, p-mTOR/mTOR, and HIF-1α. Compared with the normal group, the OGD group exhibited a significant decrease in cell viability(P<0.01), an increase in autophagosomes(P<0.01), enhanced fluorescence intensity of autophagosomes(P<0.01), up-regulated Beclin1, LC3-Ⅱ/LC3-Ⅰ, and HIF-1α(P<0.05 or P<0.01), and down-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.05 or P<0.01). Compared with the OGD group, the low-and medium-dose AS-Ⅳ groups and the DEX group showed a significant increase in cell viability(P<0.01), decreased autophagosomes(P<0.01), weakened fluorescence intensity of autophagosomes(P<0.01), down-regulated Beclin1, LC3-Ⅱ/LC3-Ⅰ, and HIF-1α(P<0.05 or P<0.01), and up-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.01). AS-Ⅳ at low and medium doses exerted a protective effect against OGD-induced autophagic injury in PC12 cells by activating the Akt/mTOR pathway, subsequently influencing HIF-1α. The high-dose AS-Ⅳ group did not show a statistically significant difference compared with the OGD group. This study provides a certain target reference for the prevention and treatment of OGD-induced cellular autophagic injury by AS-Ⅳ and accumulates laboratory data for the secondary development of Astragali Radix and AS-Ⅳ.
Rats ; Animals ; PC12 Cells ; Proto-Oncogene Proteins c-akt/genetics* ; Glucose/therapeutic use* ; Oxygen/metabolism* ; Beclin-1/pharmacology* ; TOR Serine-Threonine Kinases/metabolism* ; Autophagy ; Apoptosis ; Reperfusion Injury/drug therapy*

This study aims to investigate the effect and mechanism of saikosaponin D on the proliferation, apoptosis, and autophagy of pancreatic cancer Panc-1 cells. The cell counting kit(CCK-8) was used to examine the effects of 7, 10, 13, 16, 19, 22, 25, and 28 μmol·L~(-1) saikosaponin D on the proliferation of Panc-1 cells. Three groups including the control(0 μmol·L~(-1)), low-concentration(10 μmol·L~(-1)) saikosaponin D, and high-concentration(16 μmol·L~(-1)) saikosaponin D groups were designed. The colony formation assay was employed to measure the effect of saikosaponin D on the colony formation rate of Panc-1 cells. The cells treated with saikosaponin D were stained with hematoxylin-eosin(HE), and the changes of cell morphology were observed. Hoechst 33258 fluorescent staining was used to detect the effect of saikosaponin D on the cell apoptosis. The autophagy staining assay kit with MDC was used to examine the effect of saikosaponin D on the autophagy of Panc-1 cells. Western blot and immunocytochemistry(ICC) were employed to examine the effect of saikosaponin D on the expression levels and distribution of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cysteine-aspartic acid protease-3(caspase-3), cleaved caspase-3, autophagy-associated protein Beclin1, microtubule-associated protein light chain 3(LC3), protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), mammalian target of rapamycin(mTOR), and phosphorylated mammalian target of rapamycin(p-mTOR). The results showed that compared with the control group, saikosaponin D decreased the proliferation rate of Panc-1 cells in a dose-dependent and time-dependent manner. The colony formation rate of the cells significantly decreased after saikosaponin D treatment. Compared with the control group, the cells treated with saikosaponin D became small, accompanied by the formation of apoptotic bodies. The saikosaponin D groups showed increased apoptosis rate and autophagic vesicle accumulation. Compared with the control group, saikosaponin D up-regulated the expression of Bax, cleaved caspase3, Beclin1, LC3Ⅱ/LC3Ⅰ and down-regulated the expression of Bcl-2, caspase-3, p-Akt/Akt, and p-mTOR/mTOR. In addition, these proteins mainly existed in the cytoplasm. In conclusion, saikosaponin D can inhibit the proliferation and induce the apoptosis and autophagy of Panc-1 cells via inhibiting the Akt/mTOR pathway.
Humans ; Proto-Oncogene Proteins c-akt/genetics* ; Caspase 3 ; bcl-2-Associated X Protein ; Beclin-1/pharmacology* ; Cell Line, Tumor ; TOR Serine-Threonine Kinases/genetics* ; Apoptosis ; Pancreatic Neoplasms/drug therapy* ; Caspases ; Autophagy

This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen. H9c2 cells were assigned into five groups: control(CON) group, intermittent low oxygen(IH) group, intermittent low oxygen plus DBD-containing serum(IH+DBD) group, intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group, and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group. Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes. Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells, and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture, which would reflect the degree of cell damage. TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells, and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential. Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ), microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ), P62, Parkin and apoptosis related proteins pro caspase-3, caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax). The results showed that compared with the CON group, the IH group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated expression of P62. In addition, the IH group showed decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. Compared with the IH group, the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining, increased LC3Ⅱ/LC3Ⅰ ratio, up-regulated Parkin expression, and down-regulated P62 expression. In addition, the two groups showed increased cell survival rate, reduced content of LDH and CK in the culture medium, decreased number of TUNEL positive cells, and increased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. The IH+DBD+3-MA and IH groups showed no significant differences in the above indicators. Compared with the IH+DBD group, the IH+DBD+3-MA group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated P62 expression. In addition, the group had decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios, and declined mitochon-drial membrane potential. To sum up, DBD could promote the mitophagy, inhibit the apoptosis, and alleviated the injury of H9c2 cells exposed to low oxygen.
Oxygen ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/genetics* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Autophagy ; Ubiquitin-Protein Ligases ; Microtubule-Associated Proteins

OBJECTIVES: Hyperhomocysteinaemia (Hcy) is an independent risk factor for cardiovascular and cerebrovascular diseases. MicroRNA (miR)-18a-5p is closely related to cardiovascular diseases. This study aims to investigate the effects of miR-18a-5p on homocysteine (Hcy)-induced myocardial cells injury. METHODS: H9c2 cells were transfected with miR-18a-5p mimic/miR-18a-5p mimic negative control (NC) or combined with Hcy for intervention, and untreated cells were set as a control group. The transfection efficiency was verified by real-time RT-PCR, and cell counting kit-8 (CCK-8) assay was used to determine cell viability. Flow cytometry was used to detect apoptosis and reactive oxygen species (ROS) levels. Western blotting was performed to measure the protein levels of microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin1, p62, Bax, Bcl-2, and Notch2. Dual luciferase reporter assay was used to detect the interaction of miR-18a-5p with Notch2. RESULTS: Compared with the control, treatment with Hcy or transfection with miR-18a-5p mimic alone, or combined treatment with Hcy and miR-18a-5p mimic/miR-18a-5p mimic NC significantly reduced the H9c2 cell viability, promoted apoptosis and ROS production, up-regulated the expressions of Bax and Beclin, down-regulated the expressions of Bcl-2, p62, and Notch2, and increased the ratio of LC3-II/LC3-I (all P<0.05). Compared with the combined intervention of miR-18a-5p mimic NC and Hcy group, the above indexes were more significantly changed in the combined intervention of miR-18a-5p mimic and Hcy group, and the difference between the 2 groups was statistically significant (all P<0.05). There is a targeted binding between Notch2 and miR-18a-5p. CONCLUSIONS MiR-18a-5p could induce autophagy and apoptosis via increasing ROS production in cardiomyocytes, and aggravate Hcy-induced myocardial injury. Notch2 is a target of miR-18a-5p.
Apoptosis/genetics* ; Autophagy/genetics* ; bcl-2-Associated X Protein ; MicroRNAs/metabolism* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; Reactive Oxygen Species ; Rats ; Animals ; Myocytes, Cardiac/drug effects* ; Homocysteine/adverse effects* ; Hyperhomocysteinemia

OBJECTIVE: To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection. METHODS: The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs. RESULTS: A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated (P < 0.01). CONCLUSION DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.
Humans ; Autophagy ; Cell Death ; Cell Nucleus ; Human Umbilical Vein Endothelial Cells/virology* ; Dengue ; Proteome