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

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

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 investigate the effects of tanshinone IIA on apoptosis and autophagy induced by hypoxia/reoxygenation in H9C2 cardiomyocytes and its mechanism. METHODS: H9C2 cardiomyocytes in logarithmic growth phase were divided into control group, hypoxia/reoxygenation model group and tanshinone IIA low-dose, medium-dose and high-dose groups (50, 100, 200 mg/L tanshinone IIA were treated after hypoxia/reoxygenation respectively). The dose with good therapeutic effect was selected for follow-up study. The cells were divided into control group, hypoxia/reoxygenation model group, tanshinone IIA+pcDNA3.1-NC group and tanshinone IIA+pcDNA3.1-ABCE1 group. The cells were transfected with the overexpressed plasmids pcDNA3.1-ABCE1 and pcDNA3.1-NC and then treated accordingly. Cell counting kit-8 (CCK-8) was used to detect H9C2 cell activity in each group. The apoptosis rate of cardiomyocytes was detected by flow cytometry. The ATP-binding cassette transporter E1 (ABCE1), apoptosis-related proteins Bcl-2 and Bax, caspase-3, autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3II/I) and p62 mRNA expression level of H9C2 cells in each group were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The protein expression levels of the above indexes in H9C2 cells were detected by Western blotting. RESULTS: (1) Cell activity and ABCE1 expression: tanshinone IIA inhibited the activity of H9C2 cells induced by hypoxia/reoxygenation, and the effect was significant at medium-dose [(0.95±0.05)% vs. (0.37±0.10)%, P < 0.01], mRNA and protein expression of ABCE1 were significantly reduced [ABCE1 mRNA (2^-ΔΔCt): 2.02±0.13 vs. 3.74±0.17, ABCE1 protein (ABCE1/GAPDH): 0.46±0.04 vs. 0.68±0.07, both P < 0.05]. (2) Expression of apoptosis-related proteins: medium-dose of tanshinone IIA inhibited the apoptosis of H9C2 cells induced by hypoxia/reoxygenation [apoptosis rate: (28.26±2.52)% vs. (45.27±3.07)%, P < 0.05]. Compared with the hypoxia/reoxygenation model group, medium-dose of tanshinone IIA significantly down-regulated the protein expression of Bax and caspase-3 in H9C2 cells induced by hypoxia/reoxygenation, and significantly up-regulated the protein expression of Bcl-2 [Bax (Bax/GAPDH): 0.28±0.03 vs. 0.47±0.03, caspase-3 (caspase-3/GAPDH): 0.31±0.02 vs. 0.44±0.03, Bcl-2 (Bcl-2/GAPDH): 0.53±0.02 vs. 0.37±0.05, all P < 0.05]. (3) Expression of autophagy-related proteins: compared with the control group, the positive rate of LC3 in the hypoxia/reoxygenation model group was significantly increased, while the positive rate of LC3 in the medium-dose of tanshinone IIA group was significantly decreased [(20.67±3.09)% vs. (42.67±3.86)%, P < 0.01]. Compared with hypoxia/reoxygenation model group, medium-dose of tanshinone IIA significantly down-regulated Beclin-1, LC3II/I and p62 protein expressions [Beclin-1 (Beclin-1/GAPDH): 0.27±0.05 vs. 0.47±0.03, LC3II/I ratio: 0.24±0.05 vs. 0.47±0.04, p62 (p62/GAPDH): 0.21±0.03 vs. 0.48±0.02, all P < 0.05]. (4) Expression of apoptosis and autophagy related proteins after transfection with overexpressed ABCE1 plasmid: compared with tanshinone IIA+pcDNA3.1-NC group, the protein expression levels of Bax, caspase-3, Beclin-1, LC3II/I and p62 in tanshinone IIA+pcDNA3.1-ABCE1 group were significantly up-regulated, while the protein expression level of Bcl-2 was significantly down-regulated. CONCLUSIONS 100 mg/L tanshinone IIA could inhibit autophagy and apoptosis of cardiomyocytes by regulating the expression level of ABCE1. So, it protects H9C2 cardiomyocytes injury induced by hypoxia/reoxygenation.
Humans ; Apoptosis ; ATP-Binding Cassette Transporters/metabolism* ; Autophagy ; bcl-2-Associated X Protein/metabolism* ; Beclin-1/metabolism* ; Caspase 3/metabolism* ; Follow-Up Studies ; Myocytes, Cardiac ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger/metabolism* ; Cell Hypoxia

OBJECTIVE: To investigate whether hydrogen-rich water exerts a protective effect against cellular injury by affecting the level of autophagy after oxygen glucose deprivation/reoxygenation (OGD/R) in a mouse hippocampal neuronal cell line (HT22 cells). METHODS: HT22 cells in logarithmic growth phase were cultured in vitro. Cell viability was detected by cell counting kit-8 (CCK-8) assay to find the optimal concentration of Na₂S₂O₄. HT22 cells were divided into control group (NC group), OGD/R group (sugar-free medium+10 mmol/L Na₂S₂O₄ treated for 90 minutes and then changed to normal medium for 4 hours) and hydrogen-rich water treatment group (HW group, sugar-free medium+10 mmol/L Na₂S₂O₄ treated for 90 minutes and then changed to medium containing hydrogen-rich water for 4 hours). The morphology of HT22 cells was observed by inverted microscopy; cell activity was detected by CCK-8 method; cell ultrastructure was observed by transmission electron microscopy; the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 was detected by immunofluorescence; the protein expression of LC3II/I and Beclin-1, markers of cellular autophagy, was detected by Western blotting. RESULTS: Inverted microscopy showed that compared with the NC group, the OGD/R group had poor cell status, swollen cytosol, visible cell lysis fragments and significantly lower cell activity [(49.1±2.7)% vs. (100.0±9.7)%, P < 0.01]; compared with the OGD/R group, the HW group had improved cell status and remarkably higher cell activity [(63.3±1.8)% vs. (49.1±2.7)%, P < 0.01]. Transmission electron microscopy showed that the neuronal nuclear membrane of cells in the OGD/R group was lysed and a higher number of autophagic lysosomes were visible compared with the NC group; compared with the OGD/R group, the neuronal damage of cells in the HW group was reduced and the number of autophagic lysosomes was notably decreased. The results of immunofluorescence assay showed that the expressions of LC3 and Beclin-1 were outstandingly enhanced in the OGD/R group compared with the NC group, and the expressions of LC3 and Beclin-1 were markedly weakened in the HW group compared with the OGD/R group. Western blotting assay showed that the expressions were prominently higher in both LC3II/I and Beclin-1 in the OGD/R group compared with the NC group (LC3II/I: 1.44±0.05 vs. 0.37±0.03, Beclin-1/β-actin: 1.00±0.02 vs. 0.64±0.01, both P < 0.01); compared with the OGD/R group, the protein expression of both LC3II/I and Beclin-1 in the HW group cells were notably lower (LC3II/I: 0.54±0.02 vs. 1.44±0.05, Beclin-1/β-actin: 0.83±0.07 vs. 1.00±0.02, both P < 0.01). CONCLUSIONS Hydrogen-rich water has a significant protective effect on OGD/R-causing HT22 cell injury, and the mechanism may be related to the inhibition of autophagy.
Mice ; Animals ; Oxygen/metabolism* ; Beclin-1/pharmacology* ; Glucose/metabolism* ; Actins ; Sincalide ; Autophagy/physiology* ; Hydrogen/pharmacology* ; Reperfusion Injury ; Apoptosis

OBJECTIVES: To explore the effect mechanism of electroacupuncture (EA) on functional constipation (FC) at the combined lower he-sea and front-mu points of large intestine based on enteric neuronal autophagy. METHODS: A total of 40 SPF Kunming mice were randomly divided into 5 groups (n = 8), i.e. a control group, a model group, an acupuncture group, a 3-methyl adenine (3-MA) group, and a 3-MA + acupuncture group. Except the control group, the FC model was established by gavage with compound diphenoxylate suspension for 14 days in the other 4 groups. After successful modeling, the mice of the acupuncture group and the 3-MA + acupuncture group received EA at bilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37), stimulated for 30 min with disperse-dense wave, 2 Hz/15 Hz of frequency, 1 mA of intensity. EA was delivered once daily. One course of treatment was composed of 5 days and 2 courses were needed, with an interval of 2 days. An intraperitoneal injection of 3-MA (15 mg/kg) was administered 30 min before EA in the mice of the 3-MA group and the 3-MA + acupuncture group, once daily. Before and after intervention, the time of the first black stool defecation and defecation behaviors in 6 h were observed in each group. After intervention, in every group, the small intestine propulsion rate was calculated, the colon tissue morphology was observed using HE staining, the ultrastructure of enteric neuronal autophagy was observed under transmission electron microscope, and the expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1 and neuronal nuclear antigen protein (NeuN) in neurons of colonic muscularis were determined by immunohistochemistry. RESULTS: Before intervention, when compared with those in the control group, the time of the first black stool defecation was prolonged (P<0.01, P<0.05), and numbers (P<0.01), wet weight (P<0.01, P<0.05) and water content (P<0.05, P<0.01) of stool in 6 h were reduced in the model, acupuncture, 3-MA and 3-MA + acupuncture groups. After intervention, compared with those in the control group, the time of the first black stool defecation was longer (P<0.05), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were decreased in the model group. The time of the first black stool defecation was shortened (P<0.01), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were increased in the acupuncture group when compared with those in the model group. The time of the first black stool defecation was extended (P<0.01), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were declined in the 3-MA + acupuncture group in comparison with those in the acupuncture group. All layers of colon tissue were normal and intact in each group. When compared with the control group, the small intestine propulsion rate and the average optical density (OD) values of LC3, Beclin-1 and NeuN in neurons of colonic muscularis were decreased (P<0.01), and autophagosomes were dropped in the model group. In the acupuncture group, the small intestine propulsion rate and the average OD values of NeuN, LC3 and Beclin-1 in neurons of colonic muscularis increased (P<0.01,P<0.05), and autophagosomes were elevated when compared with those in the model group. The small intestine propulsion rate and the average OD values of NeuN, LC3 and Beclin-1 in neurons of colonic muscularis were dropped (P<0.05,P<0.01) in the 3-MA + acupuncture group in comparison with those in the acupuncture group. CONCLUSIONS Electroacupuncture may promote enteric neuronal autophagy and increase the number of neurons so that the intestinal motility can be improved and constipation symptoms can be relieved in FC mice.
Mice ; Animals ; Electroacupuncture ; Beclin-1 ; Acupuncture Points ; Constipation/therapy* ; Intestine, Small ; Autophagy ; Water

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