OBJECTIVES: To investigate the antioxidative mechanism of snake venom-derived protein C activator (PCA) in mitigating vascular endothelial cell injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM containing 1.0 g/L D-glucose and exposed to hypoxia (1% O₂) for 6 h followed by reoxygenation for 2 h to establish a cell model of oxygen-glucose deprivation/reoxygenation (OGD/R). The cell model was treated with 2 μg/mL PCA alone or in combination with 2-ME2 (a HIF-1α inhibitor) or DMOG (a HIF-1α stabilizer), and intracellular production of reactive oxygen species (ROS) and protein expression levels of HIF-1α, BNIP3, and Beclin-1 were detected using DCFH-DA fluorescence probe, flow cytometry, and Western blotting. The OGD/R cell model was transfected with a BNIP3-specific siRNA or a scrambled control sequence prior to PCA treatment, and the changes in protein expressions of HIF-1α, BNIP3 and Beclin-1 and intracellular ROS production were examined. RESULTS: In the OGD/R cell model, PCA treatment significantly upregulated HIF-1α, BNIP3 and Beclin-1 expressions and reduced ROS production. The effects of PCA were obviously attenuated by co-treatment with 2-ME2 but augmented by treatment with DMOG (a HIF-1α stabilizer). In the cell model with BNIP3 knockdown, PCA treatment increased BNIP3 expression and decreased ROS production without causing significant changes in HIF-1α expression. Compared with HUVECs with PCA treatment only, the cells with BNIP3 knockdown prior to PCA treatment showed significantly lower Beclin-1 expression and higher ROS levels. CONCLUSIONS Snake venom PCA alleviates OGD/R-induced endothelial cell injury by upregulating HIF-1α/BNIP3 signaling to suppress ROS generation, suggesting its potential as a therapeutic agent against oxidative stress in vascular pathologies.
Humans ; Reactive Oxygen Species/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Human Umbilical Vein Endothelial Cells/drug effects* ; Membrane Proteins/metabolism* ; Proto-Oncogene Proteins/metabolism* ; Up-Regulation ; Cell Hypoxia ; Cells, Cultured ; Snake Venoms/chemistry* ; Beclin-1

OBJECTIVES: To explore the mechanism of Pingchuanning Formula (PCN) for inhibiting airway inflammation in rats with asthmatic cold syndrome. METHODS: A total of 105 SD rats were randomized equally into 7 groups, including a control group, an asthmatic cold syndrome model group, 3 PCN treatment groups at high, medium and low doses, a Guilong Kechuanning (GLCKN) treatment group, and a dexamethasone (DEX) treatment group. In all but the control rats, asthma cold syndrome models were established and daily gavage of saline, PCN, GLCKN or DEX was administered 29 days after the start of modeling. The changes in general condition, lung function and lung histopathology of the rats were observed, and inflammatory factors in the alveolar lavage fluid (BALF), oxidative stress, lung tissue ultrastructure, cytokine levels, and expressions of the genes related to the HMGB1/Beclin-1 axis and autophagy were analyzed. RESULTS: The rat models had obvious manifestations of asthmatic cold syndrome with significantly decreased body mass, food intake, and water intake, reduced FEV_0.3, FVC, and FEV_0.3/FVC, obvious inflammatory cell infiltration in the lung tissue, and increased alveolar inflammation score and counts of neutrophils, eosinophils, lymphocytes, macrophages, and leukocytes in the BALF. The rat models also had significantly increased MDA level and decreased SOD level and exhibited obvious ultrastructural changes in the lung tissues, where the expressions of HMGB1, Beclin-1, ATG5, TNF-α, IL-6,IL-1β, and IL-13 and the LC3II/I ratio were increased, while the levels of Bcl-2 and IFN-γ were decreased. PCN treatment significantly improved these pathological changes in the rat models, and its therapeutic effect was better than that of GLKCN and similar to that of DEX. CONCLUSIONS PCN can effectively alleviate airway inflammation in rat models of asthmatic cold syndrome possibly by modulating the HMGB1/Beclin-1 signaling axis to suppress cell autophagy, thereby attenuating airway inflammatory damages.
Animals ; Rats ; Autophagy/drug effects* ; Rats, Sprague-Dawley ; Asthma/pathology* ; Beclin-1 ; HMGB1 Protein/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Disease Models, Animal ; Male ; Lung/pathology* ; Inflammation

BACKGROUND: Salvianolate is a compound mainly composed of salvia magnesium acetate, which is extracted from the Chinese herb Salvia miltiorrhiza . In recent years, salvianolate injection has been widely used in the treatment of cardiovascular diseases, but the mechanism of how it can alleviate cardiotoxicity remains unclear. METHODS: The cardiac injury model was constructed by treatment with doxorubicin (Dox) or azithromycin (Azi) in zebrafish larvae. Heart phenotype, heart rate, and cardiomyocyte apoptosis were observed in the study. RNA-sequencing (RNA-seq) analysis was used to explore the underlying mechanism of salvianolate treatment. Moreover, cardiomyocyte autophagy was assessed by in situ imaging. In addition, the miR-30a/becn1 axis regulation by salvianolate was further investigated. RESULTS: Salvianolate treatment reduced the proportion of pericardial edema, recovered heart rate, and inhibited cardiomyocyte apoptosis in Dox/Azi-administered zebrafish larvae. Mechanistically, salvianolate regulated the lysosomal pathway and promoted autophagic flux in zebrafish cardiomyocytes. The expression level of becn1 was increased in Dox-induced myocardial tissue injury after salvianolate administration; overexpression of becn1 in cardiomyocytes alleviated the Dox/Azi-induced cardiac injury and promoted autophagic flux in cardiomyocytes, while becn1 knockdown blocked the effects of salvianolate. In addition, miR-30a, negatively regulated by salvianolate, partially inhibited the cardiac amelioration of salvianolate by targeting becn1  directly. CONCLUSION This study has proved that salvianolate reduces cardiomyopathy by regulating autophagic flux through the miR-30a/becn1 axis in zebrafish and is a potential drug for adjunctive Dox/Azi therapy.
Animals ; Zebrafish ; MicroRNAs/genetics* ; Autophagy/drug effects* ; Myocytes, Cardiac/metabolism* ; Cardiomyopathies/metabolism* ; Beclin-1/genetics* ; Apoptosis/drug effects* ; Plant Extracts/therapeutic use* ; Doxorubicin

OBJECTIVE: To investigate the mechanism of autophagy in regulating bacterial translocation in intestinal infection caused by hypervirulent Klebsiella pneumonia (hvKp) and explore the method of reducing translocation infection of intestinal bacteria. METHODS: Fifty C57BL/6J mice were divided into gavage group (n = 40) and control group (CO group, n = 10). The gavage group was orally administered with 200 μL/d of hvKp (colony count of 10⁹ CFU/mL) continuously for 5 days to establish a hvKp intestinal infection model. CO group was given an equal amount of normal saline. After the experiment, the mice were anesthetized with lsofluraneand euthanized with cervical dislocation under anesthesia. Peripheral venous blood of mice was collected to detect bacterial translocation by 16S rDNA sequencing, then divided into translocation group (BT+ group) and non-translocation group (BT- group). Hematoxylin-eosin (HE) staining was used to evaluate intestinal morphology. The ultrastructural changes of intestinal tissues were observed by electron microscope. The levels of intestinal oxidative stress indicators such as superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GPx) were measured. Translocation was detected by in situ hybridization. The expression of tight junction protein microtubule-associated protein 1 light chain 3-II (LC3-II) and autophagy protein Beclin-1 were measured by Western blotting. The mRNA expression of tight junction proteins ZO-1 and Claudin-2 were detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of autophagy protein and tight junction protein were observed by immunofluorescence. RESULTS: Two out of 40 mice in the gavage group died after developing aspiration pneumonia. All mice in the CO group survived. The 16S rDNA sequencing results showed that no bacteria were detected in the peripheral blood of the CO group, but bacteria were detected in the peripheral blood of 18 mice in the gavage group, with a bacterial translocation rate of 47.4%. The BT- and BT+ groups showed intestinal mucosal tissue damage, with severe damage in the BT+ group. Compared with the CO group, the level of MDA in the BT- and BT+ groups were significantly increased, while the activities of SOD and GPx were significantly decreased. Compared with the BT- group, the MDA level in the BT+ group further increased, while the SOD and GPx activities further decreased [MDA (mmol/mg): 2.98±0.11 vs. 2.48±0.11, SOD (U/mg): 62.40±5.45 vs. 73.40±4.08, GPx (U/mg): 254.72±10.80 vs. 303.55±8.57, all P < 0.01]. The results of in situ hybridization detection showed that after continuous gastric lavage for 5 days, displaced hvKp was detected in the intestinal mucosal lamina propria and liver tissue of the BT+ group. Compared with the CO group, the protein expressions of LC3-II and Beclin-1 in the BT- and BT+ groups were significantly increased. The protein expressions of LC3-II and Beclin-1 in the BT+ group were obviously lower than those in the BT- group (LC3-II/β-actin: 0.38±0.04 vs. 0.70±0.09, Beclin-1/β-actin: 0.62±0.05 vs. 0.86±0.05, both P < 0.01), and there were autophagosomes in the intestinal mucosa. These results indicated that intestinal mucosal autophagy was activated after hvKp continuous gavage. Compared with CO group, the mRNA expressions of ZO-1 and Claudin-2 in the BT- and BT+ groups were significantly decreased. Compared with the BT- group, the mRNA expressions of ZO-1 and Claudin-2 in the BT+ group was further reduced [ZO-1 mRNA (2^-ΔΔCT): 0.78±0.06 vs. 0.88±0.06, Claudin-2 mRNA (2^-ΔΔCT): 0.40±0.04 vs. 0.70±0.06, both P < 0.01]. The immunofluorescence results showed that the fluorescence intensity of LC3-II, Beclin-1, ZO-1, and Claudin-2 in the BT+ group was significantly lower than that in the BT- group. CONCLUSION HvKp can activate intestinal mucosal autophagy and reduce the damage to intestinal mucosal barrier function by down-regulating oxidative stress level, reduce the occurrence of bacterial translocation.
Animals ; Oxidative Stress ; Mice, Inbred C57BL ; Autophagy ; Intestinal Mucosa/microbiology* ; Bacterial Translocation ; Mice ; Klebsiella Infections/microbiology* ; Superoxide Dismutase/metabolism* ; Beclin-1

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*

OBJECTIVE: To explore pro-oxidative state of rotator cuff tissue and expression levels of Beclin-1 and mam-malian target of rapamycin(mTOR) in patients with acute and chronic rotator cuff injury, and then analyzed relationship between rotator cuff injury and oxidative stress and autophagy. METHODS: Forty patients with rotator cuff injury were seleceted from July 2019 to December 2020, and divided into male chronic injury group, male acute injury group, female chronic injury group, and female acute injury group, 10 patients in each group. All patients were performed rotator cuff repair under arthroscopy. The sample of tendon at the rotator cuff injury site of the patient was taken during operation, and total reactive oxygen species (ROS) and superoxide dismutase(SOD) were detected by detection kit;expression of Beclin-1 and mTOR mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR), and Western-blot was applied to detect protein expression of Beclin-1 and p-mTOR/mTOR. RESULTS: There were no significant difference in expression of ROS, SOD, Beclin-1mRNA and mTOR mRNA between male and female chronic injury groups, and between male and female acute injury groups (P>0.05); ROS, SOD and Beclin-1mRNA in male chronic injury group were higher than those in male chronic injury group, while mTOR mRNAand protein decreased (P<0.05);ROS, SOD and Beclin-1 mRNA in female chronic injury group were up-regulated compared with female acute injury group, while mTOR mRNA was down-regulated (P<0.05). CONCLUSION Chronic rotator cuff injury is more likely to stimulate the pro-oxidation state of rotator cuff tissue than acute rotator cuff injury, which could up-regulating expression of autophagy factor Beclin-1 and down-regulating expression of mTOR. Therefore, patients with chronic rotator cuff injury may have higher levels of oxidative stress and autophagy.
Female ; Humans ; Male ; Beclin-1/metabolism* ; Reactive Oxygen Species/metabolism* ; RNA, Messenger/metabolism* ; Rotator Cuff/surgery* ; Rotator Cuff Injuries/surgery* ; Superoxide Dismutase/metabolism* ; TOR Serine-Threonine Kinases/metabolism*

OBJECTIVES: This work aimed to investigate the molecular mechanism of cyclic tensile stress (CTS) stimulating autophagy in human periodontal ligament cells (hPDLCs). METHODS: hPDLCs were isolated and cultured from normal periodontal tissues. hPDLCs were loaded with tensile stress by force four-point bending extender to simulate the autophagy of hPDLCs induced by orthodontic force du-ring orthodontic tooth movement. XMU-MP-1 was used to inhibit the Hippo signaling pathway to explore the role of the Hippo-YAP signaling pathway in activating hPDLC autophagy by tensile stress. The expression levels of autophagy-related genes (Beclin-1, LC3, and p62) in hPDLCs were detected by real-time quantitative polymerase chain reaction. Western blot was used to detect the expression levels of autophagy-related proteins (Beclin-1, LC3-Ⅱ/LC3-Ⅰ, and p62) and Hippo-YAP pathway proteins (active-YAP and p-YAP) in hPDLCs. Immunofluorescence was used to locate autophagy-related proteins (LC3-Ⅱand p62) and Hippo-YAP pathway proteins (active-YAP) of hPDLCs. RESULTS: CTS-activated autophagy in hPDLCs and expression of autophagy-related proteins initially increased and then decreased; it began to increase at 30 min, peaked at 3 h, and decreased (P<0.05). CTS increased the expression of active-YAP protein and decreased the expression of p-YAP protein (P<0.05). When XMU-MP-1 inhibited the Hippo-YAP signaling pathway (P<0.05), active-YAP protein was promoted to enter the nucleus and autophagy expression was enhanced (P<0.05). CONCLUSIONS The Hippo-YAP signaling pathway is involved in the regulation of autophagy activation in hPDLCs under CTS.
Humans ; Hippo Signaling Pathway ; Periodontal Ligament/metabolism* ; Beclin-1/metabolism* ; Cells, Cultured ; Autophagy

This study explored the molecular mechanism of acteoside against hepatoma 22(H22) tumor in mice through c-Jun N-terminal kinase(JNK) signaling pathway. H22 cells were subcutaneously inoculated in 50 male BALB/c mice, and then the model mice were classified into model group, low-dose, medium-dose, and high-dose acteoside groups, and cisplatin group. The administration lasted 2 weeks for each group(5 consecutive days/week). The general conditions of mice in each group, such as mental status, diet intake, water intake, activity, and fur were observed. The body weight, tumor volume, tumor weight, and tumor-inhibiting rate were compared before and after administration. Morphological changes of liver cancer tissues were observed based on hematoxylin and eosin(HE) staining, and the expression of phosphorylated(p)-JNK, JNK, B-cell lymphoma-2(Bcl-2), Beclin-1, and light chain 3(LC3) in each tissue was detected by immunohistochemistry and Western blot. qRT-PCR was performed to detect the mRNA expression of JNK, Bcl-2, Beclin-1, and LC3. The general conditions of mice in model and low-dose acteoside groups were poor, while the general conditions of mice in the remaining three groups were improved. The body weight of mice in medium-dose acteoside group, high-dose acteoside group, and cisplatin group was smaller than that in model group(P<0.01). The tumor volume in model group was insignificantly different from that in low-dose acteoside group, and the volume in cisplatin group showed no significant difference from that in high-dose acteoside group. Tumor volume and weight in medium-dose and high-dose acteoside groups and cisplatin group were lower than those in the model group(P<0.001). The tumor-inhibiting rates were 10.72%, 40.32%, 53.79%, and 56.44% in the low-dose, medium-dose, and high-dose acteoside groups and cisplatin group, respectively. HE staining showed gradual decrease in the count of hepatoma cells and increasing sign of cell necrosis in the acteoside and cisplatin groups, and the necrosis was particularly obvious in the high-dose acteoside group and cisplatin group. Immunohistochemical results suggested that the expression of Beclin-1, LC3, p-JNK, and JNK was up-regulated in acteoside and cisplatin groups(P<0.05). The results of immunohistochemistry, Western blot, and qRT-PCR indicated that the expression of Bcl-2 was down-regulated in the medium-dose and high-dose acteoside groups and cisplatin group(P<0.01). Western blot showed that the expression of Beclin-1, LC3, and p-JNK was up-regulated in acteoside and cisplatin groups(P<0.01), and there was no difference in the expression of JNK among groups. qRT-PCR results showed that the levels of Beclin-1 and LC3 mRNA were up-regulated in the acteoside and cisplatin groups(P<0.05), and the level of JNK mRNA was up-regulated in medium-dose and high-dose acteoside groups and cisplatin group(P<0.001). Acteoside promotes apoptosis and autophagy of H22 cells in mice hepatoma cells by up-regulating the JNK signaling pathway, thus inhibiting tumor growth.
Male ; Animals ; Mice ; Cisplatin/pharmacology* ; Carcinoma, Hepatocellular/genetics* ; MAP Kinase Signaling System ; Beclin-1 ; Apoptosis ; Liver Neoplasms/genetics* ; Necrosis ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Cell Line, Tumor ; RNA, Messenger/metabolism* ; Autophagy

This study explored the effect and underlying mechanism of Stellera chamaejasme extract(SCE) on multidrug resistance of breast cancer. The chemotherapy-sensitive breast cancer cell line MCF-7 and adriamycin(ADR)-resistant cell line MCF-7/ADR were used as experimental subjects. MTT assay was used to detect cell proliferation activity. Pi staining was used to detect the cell cycle. 4',6-Diamidino-2-phenylindole, dihydrochloride(DAPI) staining and flow cytometry were used to detect apoptosis. Dansylcadaverine(MDC) staining and GFP-LC3B-Mcherry adenovirus transfection were used to detect autophagy. The protein expression of Bcl-2, Bax, caspase-9, caspase-3, LC3B, p62, and Beclin-1 was detected by Western blot. The results showed that SCE could significantly inhibit the proliferation of both sensitive and resistant breast cancer cell lines. The drug resistance factor was 0.53, which was significantly lower than 59 of ADR. Meanwhile, the proportion of sensitive/resistant cells in the G_0/G_1 phase increased significantly after SCE treatment. In addition, DAPI staining showed that a series of apoptosis phenomena such as nuclear pyknosis, staining deepening, and nuclear fragmentation appeared in sensitive/resistant cell lines after SCE administration. Moreover, the results of flow cytometry double staining showed that the proportion of apoptotic cells in sensitive/resistant cell lines increased significantly after SCE administration. Besides, Western blot showed that the protein expression levels of caspase-3, caspase-9, and Bcl-2 significantly decreased and the expression level of Bax protein significantly increased in both breast cancer cell lines after SCE administration. Furthermore, SCE could also increase the positive fluorescent spots after MDC staining and yellow fluorescent spots after GFP-LC3B-mcherry transfection, and up-regulate the expression levels of autophagy-related proteins LC3B-Ⅱ, p62, and Beclin-1 in breast cancer cells. In summary, SCE may play the role of anti-multidrug resistance by blocking the cell cycle of breast cancer multidrug-resistant cells, blocking autophagy flow, and ultimately interfering with the apoptosis resistance of drug-resistant cells.
Humans ; Female ; Breast Neoplasms/metabolism* ; MCF-7 Cells ; Caspase 3/metabolism* ; Caspase 9/metabolism* ; Beclin-1/pharmacology* ; Apoptosis ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Cell Proliferation

Diabetic ulcer(DU) is a chronic and refractory ulcer which often occurs in the foot or lower limbs. It is a diabetic complication with high morbidity and mortality. The pathogenesis of DU is complex, and the therapies(such as debridement, flap transplantation, and application of antibiotics) are also complex and have long cycles. DU patients suffer from great economic and psychological pressure while enduring pain. Therefore, it is particularly important to promote rapid wound healing, reduce disability and mortality, protect limb function, and improve the quality of life of DU patients. By reviewing the relevant literatures, we have found that autophagy can remove DU wound pathogens, reduce wound inflammation, and accelerate ulcer wound healing and tissue repair. The main autophagy-related factors microtubule-binding light chain protein 3(LC3), autophagy-specific gene Beclin-1, and ubiquitin-binding protein p62 mediate autophagy. The traditional Chinese medicine(TCM) treatment of DU mitigates clinical symptoms, accelerates ulcer wound healing, reduces ulcer recurrence, and delays further deterioration of DU. Furthermore, under the guidance of syndrome differentiation and treatment and the overall concept, TCM treatment harmonizes yin and yang, ameliorates TCM syndrome, and treats underlying diseases, thereby curing DU from the root. Therefore, this article reviews the role of autophagy and major related factors LC3, Beclin-1, and p62 in the healing of DU wounds and the intervention of TCM, aiming to provide reference for the clinical treatment of DU wounds and subsequent in-depth studies.
Humans ; Ulcer/therapy* ; Medicine, Chinese Traditional ; Beclin-1 ; Quality of Life ; Wound Healing ; Diabetes Complications ; Autophagy ; Diabetic Foot/drug therapy* ; Diabetes Mellitus/genetics*