High-dose Vitamin C injection ameliorates against sepsis-induced myocardial injury by anti-apoptosis, anti-inflammatory and pro-autophagy through regulating MAPK, NF-κB and PI3K/AKT/mTOR signaling pathways in rats.

AIMS: This study aimed to evaluate the effects of VC on SIMI in rats. METHODS: In this study, the survival rate of high dose VC for SIMI was evaluated within 7 days. Rats were randomly assigned to three groups: Sham group, CLP group, and high dose VC (500 mg/kg i.v.) group. The animals in each group were treated with drugs for 1 day, 3 days or 5 days, respectively. Echocardiography, myocardial enzymes and HE were used to detect cardiac function. IL-1ß, IL-6, IL-10 and TNF-α) in serum were measured using ELISA kits. Western blot was used to detect proteins related to apoptosis, inflammation, autophagy, MAPK, NF-κB and PI3K/Akt/mTOR signaling pathways. RESULTS: High dose VC improved the survival rate of SIMI within 7 days. Echocardiography, HE staining and myocardial enzymes showed that high-dose VC relieved SIMI in rats in a time-dependent manner. And compared with CLP group, high-dose VC decreased the expressions of pro-apoptotic proteins, while increased the expression of anti-apoptotic protein. And compared with CLP group, high dose VC decreased phosphorylation levels of Erk1/2, P38, JNK, NF-κB and IKK α/ß in SIMI rats. High dose VC increased the expression of the protein Beclin-1 and LC3-II/LC3-I ratio, whereas decreased the expression of P62 in SIMI rats. Finally, high dose VC attenuated phosphorylation of PI3K, AKT and mTOR compared with the CLP group. SIGNIFICANCE: Our results showed that high dose VC has a good protective effect on SIMI after continuous treatment, which may be mediated by inhibiting apoptosis and inflammatory, and promoting autophagy through regulating MAPK, NF-κB and PI3K/AKT/mTOR pathway.

The mechanism of dehydroandrographolide inhibiting metastasis in gastric cancer based on network pharmacology and bioinformatics.

Gastric cancer (GC) is the most aggressive malignant tumor of the digestive tract. However, there is still a lack of effective treatment methods in clinical practice. Studies have shown that dehydroandrographolide (DA) has been shown to have anti-cancer activity in a variety of cancers, but it has not been reported in GC. Firstly, we obtained data on DA target genes, GC-related genes, and differentially expressed genes (DEGs) from the PharmMapper, GeneCards, and GEO databases, respectively. Then, the STRING database was used to construct the protein-protein interaction network of intersection genes, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of intersection genes were performed. Finally, 8 hub target genes were identified by analyzing their expression and prognostic survival, and molecular docking between the hub genes and DA was performed. In this study, 293 DA drug target genes, 11,366 GC-related genes, and 3184 DEGs were identified. Gene Ontology and KEGG analysis showed that the intersection genes of DA targets and GC-related genes were mainly related to cancer pathways involving apoptosis and cell adhesion. The intersection genes of DEGs, DA targets, and GC-related genes were also mainly related to cancer pathways involving chemical carcinogenesis, and drug metabolism. The molecular docking results showed that the 8 hub target genes had an apparent affinity for DA, which could be used as potential targets for DA treatment of GC. The results of this study show that the molecular mechanism by which DA inhibits GC metastasis involves multiple target genes. It may play an essential role in inhibiting the invasion and metastasis of GC by regulating the expression and polymorphism of hub target genes, such as MMP9, MMP12, CTSB, ESRRG, GSTA1, ADHIC, CA2, and AKR1C2.

18ß-glycyrrhetinic acid promotes gastric cancer cell autophagy and inhibits proliferation by regulating miR-328-3p/signal transducer and activator of transcription 3.

BACKGROUND: Gastric cancer (GC) is one of the most common cancer types worldwide, and its prevention and treatment methods have garnered much attention. As the active ingredient of licorice, 18ß-glycyrrhetinic acid (18ß-GRA) has a variety of pharmacological effects. The aim of this study was to explore the effective target of 18ß-GRA in the treatment of GC, in order to provide effective ideas for the clinical prevention and treatment of GC. AIM: To investigate the mechanism of 18ß-GRA in inhibiting cell proliferation and promoting autophagy flux in GC cells. METHODS: Whole transcriptomic analyses were used to analyze and screen differentially expressed microRNAs (miRNAs) in GC cells after 18ß-GRA intervention. Lentivirus-transfected GC cells and the Cell Counting Kit-8 were used to detect cell proliferation ability, cell colony formation ability was detected by the clone formation assay, and flow cytometry was used to detect the cell cycle and apoptosis. A nude mouse transplantation tumor model of GC cells was constructed to verify the effect of miR-328-3p overexpression on the tumorigenicity of GC cells. Tumor tissue morphology was observed by hematoxylin and eosin staining, and microtubule-associated protein light chain 3 (LC3) expression was detected by immunohistochemistry. TransmiR, STRING, and miRWalk databases were used to predict the relationship between miR-328-3p and signal transducer and activator of transcription 3 (STAT3)-related information. Expression of STAT3 mRNA and miR-328-3p was detected by quantitative polymerase chain reaction (qPCR) and the expression levels of STAT3, phosphorylated STAT3 (p-STAT3), and LC3 were detected by western blot analysis. The targeted relationship between miR-328-3p and STAT3 was detected using the dual-luciferase reporter gene system. AGS cells were infected with monomeric red fluorescent protein-green fluorescent protein-LC3 adenovirus double label. LC3 was labeled and autophagy flow was observed under a confocal laser microscope. RESULTS: The expression of miR-328-3p was significantly upregulated after 18ß-GRA intervention in AGS cells (P = 4.51E-06). Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation ability, arrested the cell cycle in the G0/G1 phase, promoted cell apoptosis, and inhibited the growth of subcutaneous tumors in BALB/c nude mice (P < 0.01). No obvious necrosis was observed in the tumor tissue in the negative control group (no drug intervention or lentivirus transfection) and vector group (the blank vector for lentivirus transfection), and more cells were loose and necrotic in the miR-328-3p group. Bioinformatics tools predicted that miR-328-3p has a targeting relationship with STAT3, and STAT3 was closely related to autophagy markers such as p62. After overexpressing miR-328-3p, the expression level of STAT3 mRNA was significantly decreased (P < 0.01) and p-STAT3 was downregulated (P < 0.05). The dual-luciferase reporter gene assay showed that the luciferase activity of miR-328-3p and STAT3 3' untranslated regions of the wild-type reporter vector group was significantly decreased (P < 0.001). Overexpressed miR-328-3p combined with bafilomycin A1 (Baf A1) was used to detect the expression of LC3 II. Compared with the vector group, the expression level of LC3 II in the overexpressed miR-328-3p group was downregulated (P < 0.05), and compared with the Baf A1 group, the expression level of LC3 II in the overexpressed miR-328-3p + Baf A1 group was upregulated (P < 0.01). The expression of LC3 II was detected after intervention of 18ß-GRA in GC cells, and the results were consistent with the results of miR-328-3p overexpression (P < 0.05). Additional studies showed that 18ß-GRA promoted autophagy flow by promoting autophagosome synthesis (P < 0.001). qPCR showed that the expression of STAT3 mRNA was downregulated after drug intervention (P < 0.05). Western blot analysis showed that the expression levels of STAT3 and p-STAT3 were significantly downregulated after drug intervention (P < 0.05). CONCLUSION: 18ß-GRA promotes the synthesis of autophagosomes and inhibits GC cell proliferation by regulating the miR-328-3p/STAT3 signaling pathway.

Xuebijing injection protects sepsis induced myocardial injury by mediating TLR4/NF-κB/IKKα and JAK2/STAT3 signaling pathways.

OBJECTIVE: Compelling evidence has demonstrated that Xuebijing (XBJ) exerted protective effects against SIMI. The aims of this study were to investigate whether TLR4/IKKα-mediated NF-κB and JAK2/STAT3 pathways were involved in XBJ's cardio-protection during sepsis and the mechanisms. METHODS: In this study, rats were randomly assigned to three groups: Sham group; CLP group; XBJ group. Rats were treated with XBJ or sanitary saline after CLP. Echocardiography, myocardial enzymes and HE were used to detect cardiac function. IL-1ß, IL-6 and TNF-α in serum were measured using ELISA kits. Cardiomyocyte apoptosis were tested by TUNEL staining. The protein levels of Bax, Bcl-2, Bcl-xl, Cleaved-Caspase 3, Cleaved-Caspase 9, Cleaved-PARP, TLR4, p-NF-κB, p-IKKα, p-JAK2 and p-STAT3 in the myocardium were assayed by western blotting. And finally, immunofluorescence was used to assess the level of p-JAK2 and p-STAT3 in heart tissue. RESULTS: The results of echocardiography, myocardial enzyme and HE test showed that XBJ could significantly improve SIMI. The IL-1ß, IL-6 and TNF-α levels in the serum were markedly lower in the XBJ group than in the CLP group (p<0.05). TUNEL staining's results showed that XBJ ameliorated CLP-induced cardiomyocyte apoptosis. Meanwhile, XBJ downregulated the protein levels of Bax, Cleaved-Caspase 3, Cleaved-Caspase 9, Cleaved-PARP, TLR4, p-NF-κB, p-IKKα, p-JAK2 and p-STAT3, as well as upregulated the protein levels of Bcl-2, Bcl-xl (p <0.05). CONCLUSIONS: In here, we observed that XBJ's cardioprotective advantages may be attributable to its ability to suppress inflammation and apoptosis via inhibiting the TLR4/ IKKα-mediated NF-κB and JAK2/STAT3 pathways during sepsis.

18ß-glycyrrhetinic acid inhibits proliferation of gastric cancer cells through regulating the miR-345-5p/TGM2 signaling pathway.

BACKGROUND: Gastric cancer (GC) is a common gastrointestinal malignancy worldwide. Based on cancer-related mortality, the current prevention and treatment strategies for GC still show poor clinical results. Therefore, it is important to find effective drug treatment targets. AIM: To explore the molecular mechanism of 18ß-glycyrrhetinic acid (18ß-GRA) regulating the miR-345-5p/TGM2 signaling pathway to inhibit the proliferation of GC cells. METHODS: CCK-8 assay was used to determine the effect of 18ß-GRA on the survival rate of GES-1 cells and AGS and HGC-27 cells. Cell cycle and apoptosis were detected by flow cytometry, cell migration was detected by a wound healing assay, the effect of 18ß-GRA on subcutaneous tumor growth in BALB/c nude mice was investigated, and the cell autophagy level was determined by MDC staining. TMT proteomic analysis was used to detect the differentially expressed autophagy-related proteins in GC cells after 18ß-GRA intervention, and then the protein-protein interaction was predicted using STRING (https://string-db.org/). MicroRNAs (miRNAs) transcriptome analysis was used to detect the miRNA differential expression profile, and use miRBase (https://www.mirbase/) and TargetScan (https://www.targetscan.org/) to predict the miRNA and complementary binding sites. Quantitative real-time polymerase chain reaction was used to detect the expression level of miRNA in 18ß-GRA treated cells, and western blot was used to detect the expression of autophagy related proteins. Finally, the effect of miR-345-5p on GC cells was verified by mir-345-5p overexpression. RESULTS: 18ß-GRA could inhibit GC cells viability, promote cell apoptosis, block cell cycle, reduce cell wound healing ability, and inhibit the GC cells growth in vivo. MDC staining results showed that 18ß-GRA could promote autophagy in GC cells. By TMT proteomic analysis and miRNAs transcriptome analysis, it was concluded that 18ß-GRA could down-regulate TGM2 expression and up-regulate miR-345-5p expression in GC cells. Subsequently, we verified that TGM2 is the target of miR-345-5p, and that overexpression of miR-345-5p significantly inhibited the protein expression level of TGM2. Western blot showed that the expression of autophagy-related proteins of TGM2 and p62 was significantly reduced, and LC3II, ULK1 and AMPK expression was significantly increased in GC cells treated with 18ß-GRA. Overexpression of miR-345-5p not only inhibited the expression of TGM2, but also inhibited the proliferation of GC cells by promoting cell apoptosis and arresting cell cycle. CONCLUSION: 18ß-GRA inhibits the proliferation of GC cells and promotes autophagy by regulating the miR-345-5p/TGM2 signaling pathway.

Xuebijing injection protects against sepsis-induced myocardial injury by regulating apoptosis and autophagy via mediation of PI3K/AKT/mTOR signaling pathway in rats.

OBJECTIVE: Apoptosis and autophagy are significant factors of sepsis induced myocardial injury (SIMI). XBJ improves SIMI by PI3K/AKT/mTOR pathway. Present study is devised to explore the protective mechanism of XBJ in continuous treatment of SIMI caused by CLP. METHODS: Rat survival was first recorded within 7 days. Rats were randomly assigned to three groups: Sham group, CLP group, and XBJ group. The animals in each group were divided into 12 h group, 1 d, 2 d, 3 d and 5 d according to the administration time of 12 hours, 1 day, 2 days, 3 days or 5 days, respectively. Echocardiography, myocardial injury markers and H&E staining were used to detect cardiac function and injury. IL-1ß, IL-6 and TNF-α in serum were measured using ELISA kits. Cardiomyocyte apoptosis was assayed by TUNEL staining. Apoptosis and autophagy related proteins regulated by the PI3K/AKT/mTOR signaling pathway were tested using western blot. RESULTS: XBJ increased the survival rate in CLP-induced septic Rat. First of all, the results of echocardiography, H&E staining and myocardial injury markers (cTnI, CK, and LDH levels) showed that XBJ could effectively improve the myocardial injury caused by CLP with the increase of treatment time. Moreover, XBJ significantly decreased the levels of serum inflammatory cytokines IL-1ß, IL-6 and TNF-α in SIMI rats. Meanwhile, XBJ downregulated the expression of apoptosis-related proteins Bax, Cleaved-Caspase 3, Cleaved-Caspase 9, Cytochrome C and Cleaved-PARP, while upregulated the protein levels of Bcl-2 in SIMI rats. And, XBJ upregulated the expression of autophagy related protein Beclin-1 and LC3-II/LC3-I ratio in SIMI rats, whereas downregulated the expression of P62. Finally, XBJ administration downregulated the phosphorylation levels of proteins PI3K, AKT and mTOR in SIMI rats. CONCLUSIONS: Our results showed that XBJ has a good protective effect on SIMI after continuous treatment, and it was speculated that it might be through inhibiting apoptosis and promoting autophagy via, at least partially, activating PI3K/AKT/mTOR pathway in the early stage of sepsis, as well as promoting apoptosis and inhibiting autophagy via suppressing PI3K/AKT/mTOR pathway in the late stage of sepsis.

Therapeutic Effect of Mongolian Medicine RuXian-I on Hyperplasia of Mammary Gland Induced by Estrogen/Progesterone through CRYAB-Promoted Apoptosis.

The traditional Mongolian medicine (TMM) RuXian-I is an empirical formula specifically used for treating the hyperplasia of mammary gland (HMG) in clinic based on the principles of traditional Mongolian medicine, but the treatment mechanism is not completely clear. In this paper, we elaborated the mechanism of RuXian-I in the treatment of HMG induced by estrogen and progestogen from its toxicity and activity. Firstly, RuXian-I exhibited no toxic effect on HMG rats through no changes of body weight and food intake measurement and no pathologic changes of the organs (heart, liver, spleen, lung, and kidney) detected. Secondly, RuXian-I could decrease the increased nipple height and diameter and remarkably relieve the pathologic changes of HMG rats and also alleviate serum sex hormone levels (estradiol (E2), luteinizing hormone (LH), progesterone (P), and testosterone (T)) of HMG rats. Finally, RuXian-I could obviously inhibit the upregulation level of antiapoptotic protein CRYAB of HMG rats and promote mammary gland cell apoptosis of HMG rats via increases of promoting apoptosis protein caspases-3, 8, and 9 and Bax and tumor suppressor protein p53, decreases of antiapoptosis protein Bcl-2, and release of cytochrome c. These results suggested that RuXian-I has protective and therapeutic effects on HMG rats induced by estrogen and progestogen possibly via promoting apoptotic pathway regulated by CRYAB and is a promising agent for treating HMG.

MAPT/Tau accumulation represses autophagy flux by disrupting IST1-regulated ESCRT-III complex formation: a vicious cycle in Alzheimer neurodegeneration.

Macroautophagy/autophagy deficit induces intracellular MAPT/tau accumulation, the hallmark pathology in Alzheimer disease (AD) and other tauopathies; however, the reverse role of MAPT accumulation in autophagy and neurodegeneration is not clear. Here, we found that overexpression of human wild-type full-length MAPT, which models MAPT pathologies as seen in sporadic AD patients, induced autophagy deficits via repression of autophagosome-lysosome fusion leading to significantly increased LC3 (microtubule-associated protein 1 light chain 3)-II and SQSTM1/p62 (sequestosome 1) protein levels with autophagosome accumulation. At the molecular level, intracellular MAPT aggregation inhibited expression of IST1 (IST1 factor associated with ESCRT-III), a positive modulator for the formation of ESCRT (the Endosomal Sorting Complex Required for Transport) complex that is required for autophagosome-lysosome fusion. Upregulating IST1 in human MAPT transgenic mice attenuated autophagy deficit with reduced MAPT aggregation and ameliorated synaptic plasticity and cognitive functions, while downregulating IST1 per se induced autophagy deficit with impaired synapse and cognitive function in naïve mice. IST1 can facilitate association of CHMP2B (charged multivesicular body protein 2B) and CHMP4B/SNF7-2 to form ESCRT-III complex, while lack of IST1 impeded the complex formation. Finally, we demonstrate that MAPT accumulation suppresses IST1 transcription with the mechanisms involving the ANP32A-regulated mask of histone acetylation. Our findings suggest that the AD-like MAPT accumulation can repress autophagosome-lysosome fusion by deregulating ANP32A-INHAT-IST1-ESCRT-III pathway, which also reveals a vicious cycle of MAPT accumulation and autophagy deficit in the chronic course of AD neurodegeneration.Abbreviations: AAV: adeno-associated virus; Aß: ß-amyloid; aCSF: artificial cerebrospinal fluid; AD: Alzheimer disease; ANP32A: acidic nuclear phosphoprotein 32 family member A; ATG: autophagy related; AVs: autophagic vacuoles; CEBPB: CCAAT enhancer binding protein beta; CHMP: charged multivesicular body protein; DMEM: Dulbecco's modified eagle's medium; EBSS: Earle's balanced salt solution; EGFR: epidermal growth factor receptor; ESCRT: endosomal sorting complex required for transport; fEPSPs: field excitatory postsynaptic potentials; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3B: glycogen synthase kinase 3 beta; HAT: histone acetyl transferase; HDAC: histone deacetylase; INHAT: inhibitor of histone acetyl transferase; IST1: IST1 factor associated with ESCRT-III; LAMP2: lysosomal associated membrane protein 2; LTP: long-term potentiation; MAP1LC3: microtubule associated protein 1 light chain 3; MAPT/tau: microtubule associated protein tau; MVB: multivesicular bodies; MWM: Morris water maze; PBS: phosphate-buffered saline solution; RAB7: member RAS oncogene family; SNAREs: soluble N-ethylmaleimide-sensitive factor attachment protein receptors; SQSTM1/p62: sequestosome 1.

Inhibition of Histone Acetylation by ANP32A Induces Memory Deficits.

There is accumulating evidence that decreased histone acetylation is involved in normal aging and neurodegenerative diseases. Recently, we found that ANP32A, a key component of INHAT (inhibitor of acetyltransferases) that suppresses histone acetylation, increased in aged and cognitively impaired C57 mice and expressing wild-type human full length tau (htau) transgenic mice. Downregulating ANP32A restored cognitive function and synaptic plasticity through upregulation of the expressions of synaptic-related proteins via increasing histone acetylation. However, there is no direct evidence that ANP32A can induce neurodegeneration and memory deficits. In the present study, we overexpressed ANP32A in the hippocampal CA3 region of C57 mice and found that ANP32A overexpression induced cognitive abilities and synaptic plasticity deficits, with decreased synaptic-related protein expression and histone acetylation. Combined with our recent studies, our findings reveal that upregulated ANP32A induced-suppressing histone acetylation may underlie the cognitive decline in neurodegenerative disease, and suppression of ANP32A may represent a promising therapeutic approach for neurodegenerative diseases including Alzheimer's disease.

Novel therapeutic strategies for patients with triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents a very heterogeneous group of breast diseases. Currently, the backbone of therapy for TNBC is mainly chemotherapy as there are no effective specific targeted agents approved to treat TNBC. Despite initial responses to chemotherapy, resistance frequently and rapidly develops and metastatic TNBC has a poor prognosis. Therefore, new targeted strategies are, accordingly, urgently needed. This article discusses the recent developments in targeted agents explored for TNBC, aiming to offer novel therapeutic strategies that can potentially assist in designing personalized therapeutics in the future as well as provide the basis for further research in an attempt to target TNBC.