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

Taking lipophagy as the breakthrough point, we explored the mechanism of Zexie Decoction(ZXD) in improving lipid metabolism in the hepatocyte model induced by palmitic acid(PA) and in the animal model induced by high-fat diet(HFD) on the basis of protein kinase B(Akt)/transcription factor EB(TFEB) signaling pathway. Co-localization was carried out for the microtubule-associated protein light chain 3(LC3) plasmid labeled with green fluorescent protein(GFP) and lipid droplets(LDs), and immunofluorescence co-localization for liver LC3 of HFD mice and perilipin 2(PLIN2). The results showed that ZXD up-regulated the expression of LC3, reduced lipid accumulation in hepatocytes, and increased the co-localization of LC3 and LDs, thereby activating lipo-phagy. Western blot results confirmed that ZXD increased autophagy-related protein LC3Ⅱ/LC3Ⅰ transformation ratio and lysosome-associated membrane protein 2(LAMP2) in vivo and in vitro and promoted the degradation of sequestosome-1(SQSTM1/p62)(P<0.05). The results above jointly explained that ZXD regulated lipophagy. Furthermore, ZXD activated TFEB expression(P<0.05) and reversed the PA-and HFD-induced decrease of TFEB nuclear localization in hepatocytes(P<0.05). Meanwhile, ZXD activated liver TFEB to up-regulate the expression of the targets Lamp2, Lc3 B, Bcl2, and Atg5(P<0.05). Additionally, ZXD down-regulated the protein level of p-Akt upstream of TFEB in vivo and in vitro. In conclusion, ZXD may promote lipophagy by regulating the Akt/TFEB pathway.
Animals ; Mice ; Autophagy/drug effects* ; Hepatocytes/metabolism* ; Microtubule-Associated Proteins/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction ; Drugs, Chinese Herbal/pharmacology*

Objective: To explore the expression of autophagy related factors microtubule associated protein 1 light chain 3B (LC3B), p62, autophagy key factor Beclin1 in oral lichen planus (OLP) tissues and their relationships with the clinicopathological characteristics of OLP, investigating the function and significance of autophagy in pathogenesis of OLP. Methods: Forty-one lesion tissues (OLP group, twenty-one cases of erosive OLP and twenty cases of non-erosive OLP) were selected from OLP patients visiting the Department of Periodontal and Oral Medicine, School and Hospital of Stomatology, Guizhou Medical University from October 2017 to December 2019. Fifteen cases of normal oral mucosal tissues (control group) were collected from oral and maxillofacial surgery at The Affiliated Stomatology Hospital of Guizhou Medical University during the same period. Protein and mRNA expression levels of LC3B, p62 and Beclin1 were detected by immunohistochemistry (IHC) and real-time quantitative PCR (RT-qPCR) in OLP lesions respectively. The protein expression levels of LC3B, p62, Beclin1 and ratio of LC3B-Ⅱ/LC3B-Ⅰ in sixteen cases (eight cases of erosive OLP and eight cases of non-erosive OLP) from the OLP group were detected by Western blotting (WB). The potential relationship between LC3B, p62, Beclin1, LC3B-Ⅱ/LC3B-Ⅰ ratio and clinical features of OLP were analyzed. Results: IHC results showed that the positive expression rates of LC3B and p62 proteins in OLP lesion tissues [LC3B: 68% (28/41); p62: 59% (24/41)] were higher than those in the control group [LC3B: 5/15; p62: 3/15] (LC3B: χ²=5.55, P=0.019; p62: χ²=5.55, P=0.015). The positive expression rates of LC3B and p62 proteins in the erosive OLP group [LC3B: 86% (18/21); p62: 76% (16/21)] were higher than those in the non-erosive OLP group [LC3B: 50% (10/20); p62: 40% (8/20)] (LC3B: χ²=4.50, P=0.034; p62:χ²=5.53, P=0.019). The positive expression rate of Beclin1 protein in the OLP lesions[20% (8/41)] was lower than that in the control group (7/15) (χ²=4.13, P=0.042), but was not statistically different between the two types of OLP (P>0.05). The RT-qPCR results showed that the mRNA expression levels of LC3B and p62 in OLP lesions [LC3B: 2.78 (1.59, 6.15); p62: 4.30 (2.34, 6.29)] were higher than those in the control group [LC3B: 1.05 (0.88, 1.21); p62: 1.12 (0.89, 1.36)] (LC3B: Z=-4.56, P<0.001; p62: Z=-4.78, P<0.001), and the mRNA expression levels of LC3B and p62 in the erosive OLP group were higher than those in the non-erosive OLP group (LC3B: Z=-2.87, P=0.004; p62: Z=-2.95, P=0.003). The mRNA expression level of Beclin1 in OLP tissues was lower than that in the control group (Z=-2.43, P=0.015), but the difference was not statistically significant between the two types of OLP (P>0.05). WB results showed that the LC3B-Ⅱ/LC3B-Ⅰ ratio was higher in the OLP lesions than that in the control group (t=-2.45, P=0.021), and the LC3B-Ⅱ/LC3B-Ⅰ ratio was higher in the non-erosive OLP group than in the erosive OLP group (t=-2.38, P=0.032). Spearman's correlation analysis showed that the ratio was negatively correlated with the clinical staging and the degree of basal cell liquefaction in OLP (clinical staging: r=-0.57, P=0.021; basal cell liquefaction: r=-0.54, P=0.032), but not with the disease duration and the degree of lymphocytic infiltration (P>0.05). Conclusions: Autophagy related factors LC3B, p62 and Beclin1 may play a role in the formation and progression of OLP lesions. The autophagy level was relatively lack in erosive OLP compared to non-erosive OLP, contributing to the increased local lesion destruction in erosive OLP. Abnormal cellular autophagy may play an important role in the formation of OLP lesions.
Humans ; Lichen Planus, Oral/metabolism* ; Beclin-1 ; Microtubule-Associated Proteins/metabolism* ; Autophagy ; RNA, Messenger/metabolism*

This study was to investigate the changes of autophagy in pancreatic tissue cells from hyperlipidemic acute pancreatitis (HLAP) rats and the molecular mechanism of autophagy to induce inflammatory injury in pancreatic tissue cells. Male Sprague Dawley (SD) rats were intraperitoneally injected with caerulein to establish acute pancreatitis (AP) model and then given a high fat diet to further prepare HLAP model. The HLAP rats were treated with autophagy inducer rapamycin or inhibitor 3-methyladenine. Pancreatic acinar (AR42J) cells were treated with caerulein to establish HLAP cell model. The HLAP cell model were treated with rapamycin or transfected with vascular endothelial growth factor (VEGF) siRNA. The inflammatory factors in serum and cell culture supernatant were detected by ELISA method. The histopathological changes of pancreatic tissue were observed by HE staining. The changes of ultrastructure and autophagy in pancreatic tissue were observed by electron microscopy. The expression levels of Beclin-1, microtubule- associated protein light chain 3-II (LC3-II), mammalian target of rapamycin complex 1 (mTORC1), and VEGF were measured by immunohistochemistry and Western blot. The results showed that, compared with control group, the autophagy levels and inflammatory injury of pancreatic tissue cells from HLAP model rats were obviously increased, and these changes were aggravated by rapamycin treatment, but alleviated by 3-methyladenine treatment. In HLAP cell model, rapamycin aggravated the autophagy levels and inflammatory injury, whereas VEGF siRNA transfection increased mTORC1 protein expression, thus alleviating the autophagy and inflammatory injury of HLAP cell model. These results suggest that VEGF-induced autophagy plays a key role in HLAP pancreatic tissue cell injury, and interference with VEGF-mTORC1 pathway can reduce the autophagy levels and alleviate the inflammatory injury. The present study provides a new target for prevention and treatment of HLAP.
Acute Disease ; Animals ; Autophagy ; Ceruletide/adverse effects* ; Male ; Mammals/metabolism* ; Mechanistic Target of Rapamycin Complex 1 ; Microtubule-Associated Proteins/metabolism* ; Pancreatitis ; RNA, Small Interfering/genetics* ; Rats ; Rats, Sprague-Dawley ; Sirolimus/adverse effects* ; Vascular Endothelial Growth Factor A/genetics*

Asthenoteratozoospermia is one of the most severe types of qualitative sperm defects. Most cases are due to mutations in genes encoding the components of sperm flagella, which have an ultrastructure similar to that of motile cilia. Coiled-coil domain containing 103 (CCDC103) is an outer dynein arm assembly factor, and pathogenic variants of CCDC103 cause primary ciliary dyskinesia (PCD). However, whether CCDC103 pathogenic variants cause severe asthenoteratozoospermia has yet to be determined. Whole-exome sequencing (WES) was performed for two individuals with nonsyndromic asthenoteratozoospermia in a consanguineous family. A homozygous CCDC103 variant segregating recessively with an infertility phenotype was identified (ENST00000035776.2, c.461A>C, p.His154Pro). CCDC103 p.His154Pro was previously reported as a high prevalence mutation causing PCD, though the reproductive phenotype of these PCD individuals is unknown. Transmission electron microscopy (TEM) of affected individuals' spermatozoa showed that the mid-piece was severely damaged with disorganized dynein arms, similar to the abnormal ultrastructure of respiratory ciliary of PCD individuals with the same mutation. Thus, our findings expand the phenotype spectrum of CCDC103 p.His154Pro as a novel pathogenic gene for nonsyndromic asthenospermia.
Asthenozoospermia/pathology* ; Dyneins/genetics* ; Homozygote ; Humans ; Male ; Microtubule-Associated Proteins ; Mutation ; Mutation, Missense ; Sperm Tail/metabolism*

The aim of the present study was to investigate the effects of dexmedetomidine (DEX) on acute liver injury induced by lipopolysaccharide (LPS)/D-galactosamine (D-Gal) and the underlying mechanism. Male BALB/c mice were intraperitoneally injected with LPS/D-Gal to induce acute liver injury model, and pretreated with DEX or in combination with the autophagy inhibitor, 3-methyladenine (3-MA) 30 min before injection. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, as well as myeloperoxidase (MPO) activity in liver tissue were determined with the corresponding kits. Serum tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) levels were determined by ELISA. The protein expression levels of LC3-II and P62 in liver tissue were determined by Western blot. Liver histopathological changes were detected by HE staining. The results showed that, compared with control group, LPS/D-Gal enhanced ALT and AST activity, increased TNF-α and IL-6 levels, as well as MPO activity, up-regulated LC3-II and P62 protein expression levels, and significantly induced pathological damage in liver tissue. DEX reversed the above changes in the LPS/D-Gal group, whereas these protective effects of DEX were blocked by 3-MA. The above results suggest that DEX alleviates LPS/D-Gal-induced acute liver injury, which may be associated with the up-regulation of LC3-II protein expression and the activation of autophagy.
Alanine Transaminase ; Animals ; Chemical and Drug Induced Liver Injury/drug therapy* ; Dexmedetomidine/pharmacology* ; Galactosamine/toxicity* ; Interleukin-6/blood* ; Lipopolysaccharides/toxicity* ; Liver ; Male ; Mice ; Mice, Inbred BALB C ; Microtubule-Associated Proteins/metabolism* ; Tumor Necrosis Factor-alpha/blood* ; Up-Regulation

We have previously reported that Cystatin C (CysC) is a pivotal mediator in the neuroprotection induced by hyperbaric oxygen (HBO) preconditioning; however, the underlying mechanism and how CysC changes after stroke are not clear. In the present study, we demonstrated that CysC expression was elevated as early as 3 h after reperfusion, and this was further enhanced by HBO preconditioning. Concurrently, LC3-II and Beclin-1, two positive-markers for autophagy induction, exhibited increases similar to CysC, while knockdown of CysC blocked these elevations. As a marker of autophagy inhibition, p62 was downregulated by HBO preconditioning and this was blocked by CysC knockdown. Besides, the beneficial effects of preserving lysosomal membrane integrity and enhancing autolysosome formation induced by HBO preconditioning were abolished in CysC rats. Furthermore, we demonstrated that exogenous CysC reduced the neurological deficits and infarct volume after brain ischemic injury, while 3-methyladenine partially reversed this neuroprotection. In the present study, we showed that CysC is biochemically and morphologically essential for promoting autophagic flux, and highlighted the translational potential of HBO preconditioning and CysC for stroke treatment.
Animals ; Autophagy ; physiology ; Beclin-1 ; metabolism ; Brain ; metabolism ; pathology ; Brain Ischemia ; metabolism ; pathology ; therapy ; Cystatin C ; genetics ; metabolism ; Disease Models, Animal ; Gene Expression ; Gene Knockdown Techniques ; Hyperbaric Oxygenation ; Lysosomes ; metabolism ; pathology ; Male ; Microtubule-Associated Proteins ; metabolism ; Neurons ; metabolism ; pathology ; Neuroprotection ; physiology ; Oxygen ; therapeutic use ; Random Allocation ; Rats, Sprague-Dawley ; Rats, Transgenic ; Reperfusion Injury ; metabolism ; pathology ; therapy

OBJECTIVE: To investigate the effects of Notch signal on hypoxic induction factor (HIF-1α) and autophagy-associated genes Beclin1, LC3I, LC3II in oxygen-glucose deprivation (OGD) induced myocardial cell injury. METHODS: The OGD model was established using hypoxic culture box and hypoglycemic DMEM medium. The cells were divided into normal control group, OGD group, OGD + NC siRNA group, OGD + Notch1 siRNA group and OGD + HIF-1α siRNA group. Western blot was used to detect the interference effects of HIF-1α siRNA and Notch1 siRNA. The effects of Notch1 siRNA and HIF-1α siRNA on the activity of myocardial cells in OGD model were detected by the CCK-8 assay. The effects of Notch1 siRNA and HIF-1α siRNA on autophage-associated genes Beclin1, LC3I and LC3II expression were detected by Western blot. RESULTS: The results of Western blot showed that HIF-1α siRNA could effectively knock down the expression of HIF-1α in myocardial cells in OGD model, and Notch1 siRNA could effectively knock down the expression of Notch1 and HIF-1α in myocardial cells in OGD model. The result of CCK-8 assay showed that Notch1 siRNA and HIF-1α siRNA reduced the activity of myocardial cells in OGD model, and there was no statistical difference between the two groups. Western blot results showed that Notch1 siRNA and HIF-1α siRNA could reduce the expressions of the autophagy-associated genes Beclin1, LC3I and LC3II, and reduce the ratio of LC3II to LC3I at mRNA level. CONCLUSION Notch1 plays a role in myocardial protection by regulating the expression of HIF-1α to regulate the autophagy in OGD model cells.
Autophagy ; Beclin-1 ; metabolism ; Cell Hypoxia ; Cells, Cultured ; Glucose ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Microtubule-Associated Proteins ; metabolism ; Myocytes, Cardiac ; cytology ; pathology ; Oxygen ; Receptors, Notch ; metabolism ; Signal Transduction

OBJECTIVE: To investigate whether autophagy mediates the effects of aldehyde dehydrogenase 2 (ALDH2) on the proliferation of neonatal rat cardiac fibroblasts cultured in high glucose. METHODS: Cardiac fibroblasts were isolated from neonatal (within 3 days) SD rats and subcultured. The fibroblasts of the third passage, after identification with immunofluorescence staining for vimentin, were treated with 5.5 mmol/L glucose (control group), 30 mmol/L glucose (high glucose group), or 30 mmol/L glucose in the presence of Alda-1 (an ALDH2 agonist), daidzin (an ALDH2 2 inhibitor), or both. Western blotting was employed to detect ALDH2, microtubule-associated protein 1 light chain 3B subunit (LC3B) and Beclin-1 in the cells, and a hydroxyproline detection kit was used for determining hydroxyproline content in cell culture medium; CCK- 8 kit was used for assessing the proliferation ability of the cardiac fibroblasts after the treatments. RESULTS: Compared with the control cells, the cells exposed to high glucose exhibited obviously decreased expressions of ALDH2, Beclin-1 and LC3B and increased cell number and hydroxyproline content in the culture medium. Treatment of the high glucose-exposed cells with Alda-1 significantly increased Beclin-1, LC3B, and ALDH2 protein expressions and lowered the cell number and intracellular hydroxyproline content, whereas the application of daidzin resulted in reverse changes in the expressions of ALDH2, Beclin-1 and LC3B, viable cell number and intracellular hydroxyproline content in high glucose-exposed cells. CONCLUSIONS Mitochondrial ALDH2 inhibits the proliferation of neonatal rat cardiac fibroblasts induced by high glucose, and the effect is possibly mediated by the up-regulation of autophagy-related proteins Beclin-1 and LC3B.
Aldehyde Dehydrogenase ; Aldehyde Dehydrogenase, Mitochondrial ; metabolism ; Animals ; Animals, Newborn ; Autophagy ; Beclin-1 ; physiology ; Fibroblasts ; Glucose ; Microtubule-Associated Proteins ; Mitochondrial Proteins ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To investigate the effect of estradiol (E2)/estrogen receptor 1 (ESR1) on the proliferation of human chondrocytes and explore the molecular mechanism. METHODS: The Ad-Easy adenovirus packaging system was used to construct and package the ESR1-overexpressing adenovirus Ad-ESR1. Western blotting and qPCR were used to detect the expression of ESR1 protein and mRNA in human chondrocyte C28I2 cells. In the cells treated with different adenoviruses, the effects of E2 were tested on the expressions of proteins related with cell autophagy and apoptosis and the phosphorylation of ERK signaling pathway using Western blotting. Immunofluorescence assay was used to observe the intracellular autophagic flow, flow cytometry was performed to analyze the cell apoptosis rate and the cell cycle changes, and qPCR was used to detect the expressions of PCNA, cyclin B1 and cyclin D1 mRNAs. The inhibitory effect of the specific inhibitor of ERK on the expressions of autophagy- and apoptosis-related genes at both the protein and mRNA levels were detected using Western blotting and qPCR. RESULTS: Transfection with the recombinant adenovirus overexpressing ESR1 and E2 treatment of C28I2 cells significantly enhanced the expressions of autophagy-related proteins LC3, ATG7, promoted the colocalization of LC3 and LAMP1 in the cytoplasm, increased the expressions of the proliferation-related marker genes PCNA, cyclin B1 and cyclin D1, and supressed the expressions of cleaved caspase-3, caspase-12 and pERK. RNA interference of ESR1 obviously lowered the expression levels of autophagy-related proteins in C28I2 cells, causing also suppression of the autophagic flow, increments of the expressions of apoptosis-related proteins and pERK, and down-regulated the expressions of the proliferation marker genes. Blocking ERK activation with the ERK inhibitor obviously inhibited the effects of E2/ESR1 on autophagy, proliferationrelated gene expressions and cell apoptosis. CONCLUSIONS The targeted binding of E2 with ESR1 promotes the proliferation of human chondrocytes possibly by inhibiting the activation of ERK signaling pathway to promote cell autophagy and induce cell apoptosis.
Adenoviridae ; metabolism ; Apoptosis ; Autophagy ; Autophagy-Related Protein 7 ; metabolism ; Cell Line ; Cell Proliferation ; Chondrocytes ; cytology ; metabolism ; Estradiol ; metabolism ; Estrogen Receptor alpha ; metabolism ; Humans ; Lysosome-Associated Membrane Glycoproteins ; metabolism ; MAP Kinase Signaling System ; Microtubule-Associated Proteins ; metabolism ; Transfection