Protective effect of water extracts of Veronicastrum latifolium (Hemsl.) Yamazaki on carbon tetrachloride-induced liver fibrosis in mice and its effect on intestinal flora.

Liver fibrosis refers to a reversible event of repair and reconstruction following injury due to various etiologies, and its continuous development will lead to cirrhosis and liver cancer. Abnormal alterations in intestinal microbiota can hasten the development of hepatic fibrosis and damage. Veronicastrum latifolium (Hemsl.) Yamazaki (VLY) is a classic drug applied extensively for managing acute and chronic hepatitis, liver cirrhosis and ascites in ethnic minority areas of Guizhou Province, China, which possesses broad-spectrum pharmacological activities. In view of the crucial role of intestinal microbiota in the development of liver fibrosis, the present study attempted to investigate the effects of VLY aqueous extract on ameliorating CCl4-elicited liver fibrosis in mice and on intestinal microbiota and to explore its possible mechanism. Phytochemical analysis showed that VLY water extract contained a variety of components, particularly rich in organic acids and their derivatives, flavonoids, phenolic acids, nucleotides and their derivatives, carbohydrates and other compounds. VLY water extract remarkably alleviated CCl4-induced liver damage and fibrosis in mice, improved liver histology, and improved liver function abnormalities. VLY water extract also inhibited the activation of hepatic stellate cells and invasion of intrahepatic inflammatory cells. Additionally, sequencing the 16 s rDNA gene revealed that VLY water extract changed the intestinal microbiota composition in liver fibrotic mice. It elevated the Firmicutes/Bacteroidota ratio and enriched the relative Lactobacillus richness, which is capable of mitigating fibrosis and inflammation in impaired liver. In summary, through modulation of inflammation and intestinal microbiota, VLY water extract can reduce the CCl4-elicited liver fibrosis.

Identification of key genes and crucial pathways for major depressive disorder using peripheral blood samples and chronic unpredictable mild stress rat models.

BACKGROUND: Accurate diagnosis of major depressive disorder (MDD) remains difficult, and one of the key challenges in diagnosing MDD is the lack of reliable diagnostic biomarkers. The objective of this study was to explore gene networks and identify potential biomarkers for MDD. METHODS: In the present study, we performed a comprehensive analysis of the mRNA expression profiles using blood samples of four patients with MDD and four controls by RNA sequencing. Differentially expressed genes (DEGs) were screened, and functional and pathway enrichment analyses were performed using the Database for Annotation, Visualization, and Integrated Discovery. All DEGs were inputted to the STRING database to build a PPI network, and the top 10 hub genes were screened using the cytoHubba plugin of the Cytoscape software. The relative expression of 10 key genes was identified by quantitative real-time polymerase chain reaction (qRT-PCR) of blood samples from 50 MDD patients and 50 controls. Plasma levels of SQSTM1 and TNFα were measured using an enzyme-linked immunosorbent assay in blood samples of 44 MDD patients and 44 controls. A sucrose preference test was used to evaluate depression-like behavior in chronic unpredictable mild stress (CUMS) model rats. Immunofluorescence assay and western blotting were performed to study the expression of proteins in the brain samples of CUMS model rats. RESULTS: We identified 247 DEGs that were closely associated with MDD. Gene ontology analyses suggested that the DEGs were mainly enriched in negative regulation of transcription by RNA polymerase II promoter, cytoplasm, and protein binding. Moreover, Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that the DEGs were significantly enriched in the MAPK signaling pathway. Ten hub genes were screened through the PPI network, and qRT-PCR assay revealed that one and six genes were downregulated and upregulated, respectively; however, SMARCA2, PPP3CB, and RAB5C were not detected. Pathway enrichment analysis for the 10 genes showed that the mTOR signaling pathway was also enriched. A strong positive correlation was observed between SQSTM1 and TNFα protein levels in patients with MDD. LC3 II and SQSTM1 protein levels were increased in the CUMS rat model; however, p-mTOR protein levels were decreased. The sucrose preference values decreased in the CUMS rat model. CONCLUSIONS: We identified 247 DEGs and constructed an MDD-specific network; thereafter, 10 hub genes were selected for further analysis. Our results provide novel insights into the pathogenesis of MDD. Moreover, SQSTM1, which is related to autophagy and inflammatory reactions, may play a key role in MDD. SQSTM1 may be used as a promising therapeutic target in MDD; additionally, more molecular mechanisms have been suggested that should be focused on in future in vivo and in vitro studies.

Increased miR­187­3p expression after cerebral ischemia/reperfusion induces apoptosis via initiation of endoplasmic reticulum stress.

Ischemia/reperfusion (I/R) injury induces activation of the endoplasmic reticulum stress (ERS) pathway, accompanied by an increase in apoptosis. Multiple microRNAs (miRNAs/miRs) are dysregulated during I/R and contribute to I/R-induced injury. miRNAs act as suppressors of gene expression and negatively regulate gene expression by targeting the protein-coding sequence (CDS) of specific target mRNAs. Seipin is an endoplasmic reticulum protein that has recently been associated with ERS. We previously reported that seipin is the target gene of miR­187­3p. Therefore, we explored the involvement of miR-187-3p in I/R-induced ERS via the regulation of seipin. A rat MCAO/R model was established by 1 h of occlusion and 24 h reperfusion. Neurological deficits and infarction area were examined. PC12 cells were exposed to oxygen­glucose deprivation/reoxygenation (OGD/R) to model I/R. Expression levels of miR-187-3p and proteins related to ERS and apoptosis were measured using RT-PCR, western blotting, immunofluorescence, and immunohistochemistry, respectively. TUNEL staining was used to assay apoptosis. MCAO/R-induced morphological changes were analyzed with Nissl staining and Hematoxylin-eosin staining. I/R-induced ERS was closely associated with an increase in miR-1873p and a decrease in seipin expression. miR-187-3p agomir further activated the ERS pathway and promoted apoptosis but decreased seipin expression levels; these effects were reversed by miR-187-3p antagomir. Moreover, seipin knockdown aggravated ERS in PC12 cells after OGD/R, and this change was rescued by seipin overexpression. miR-187-3p antagomir did not suppress ERS and apoptosis in seipin knockdown PC12 cells after OGD/R. Our findings demonstrate that the inhibition of miR­187­3p attenuated I/R­induced cerebral injury by regulating seipin-mediated ERS.

miR­187­3p inhibitor attenuates cerebral ischemia/reperfusion injury by regulating Seipin­mediated autophagic flux.

MicroRNAs (miRNAs/miRs) have been reported to affect ischemia/reperfusion (I/R)­induced cerebral damage. miRNAs cause post­transcriptional gene silencing by binding to the protein­coding sequence (CDS) of mRNAs. Seipin has a potential role in regulating autophagic flux. The present study investigated the involvement of miR­187­3p in Seipin expression, autophagic flux and apoptosis in vitro, as well as the underlying mechanism, using PC12 cells exposed to oxygen­glucose deprivation/reoxygenation (OGD/R), which mimicked the process of I/R. In comparison with control PC12 cells, OGD/R caused an increase in the level of miR­187­3p and a decrease in Seipin protein levels without changes in the level of Seipin mRNA. Using bioinformatics analysis, it was identified that miR­187­3p could bind to the CDS of Seipin. miR­187­3p inhibitor attenuated the reduction in Seipin protein expression in OGD/R­treated PC12 cells. Following OGD/R, autophagic flux was reduced and apoptosis was enhanced, which were attenuated by inhibition of miR­187­3p. Compared with OGD/R­treated PC12 cells, Seipin knockdown further impaired autophagic flux and promoted neuronal apoptosis, which were insensitive to inhibition of miR­187­3p. Furthermore, treatment with miR­187­3p inhibitor could decrease the infarction volume in a rat model of middle cerebral artery occlusion/reperfusion. The present findings indicated that miR­187­3p inhibitor attenuated ischemia­induced cerebral damage by rescuing Seipin expression to improve autophagic flux.

PNU282987 inhibits amyloid­ß aggregation by upregulating astrocytic endogenous αB­crystallin and HSP­70 via regulation of the α7AChR, PI3K/Akt/HSF­1 signaling axis.

Alzheimer's disease (AD) is a chronic and irreversible neurodegenerative disorder. Abnormal aggregation of the neurotoxic amyloid­ß (Aß) peptide is an early event in AD. The activation of astrocytic α7 nicotinic acetylcholine receptor (α7 nAChR) can inhibit Aß aggregation; thus, the molecular mechanism between α7 nAChR activation and Aß aggregation warrants further investigation. In the present study, Aß oligomer levels were assessed in astrocytic cell lysates after treatment with PNU282987 (a potent agonist of α7 nAChRs) or co­treatment with LY294002, a p­Akt inhibitor. The levels of heat shock factor­1 (HSF­1), heat shock protein 70 (HSP­70), and αB­crystallin (Cryab) in astrocytes treated with PNU282987 at various time­points or co­treated with methyllycaconitine (MLA), a selective α7 nAChR antagonist, as well as co­incubated with LY294002 were determined by western blotting. HSP­70 and Cryab levels were determined after HSF­1 knockdown (KD) in astrocytes. PNU282987 markedly inhibited Aß aggregation and upregulated HSF­1, Cryab, and HSP­70 in primary astrocytes, while the PNU282987­mediated neuroprotective effect was reversed by pre­treatment with MLA or LY294002. Moreover, the HSF­1 KD in astrocytes effectively decreased Cryab, but not HSP­70 expression. HSF­1 is necessary for the upregulation of Cryab expression, but not for that of HSP­70. HSF­1 and HSP­70 have a neuroprotective effect. Furthermore, the neuroprotective effect of PNU282987 against Aß aggregation was mediated by the canonical PI3K/Akt signaling pathway activation.

Berberine Ameliorates Oxygen-glucose Deprivation/Reperfusion-induced Apoptosis by Inhibiting Endoplasmic Reticulum Stress and Autophagy in PC12 Cells.

This study aimed to elucidate the molecular mechanisms by which berberine protects against cerebral ischemia/reperfusion (I/R) injury. The oxygen-glucose deprivation/reperfusion (OGD/R) PC12 model was established. Cell counting kit-8 (CCK-8) was used to detect the toxicity of berberine and the viability of PC12 cells. Hoechst 33258 staining and flow cytometry were used to observe the nuclear morphology, and changes of apoptosis and reactive oxygen species (ROS), respectively. Western blotting and immunofluorescence assay were employed to detect autophagy-related proteins [microtubule-associated protein 1A/1B-light chain 3 (LC3), P62/SQSTM-1, Beclin-1] and endoplasmic reticulum (ER) stress-related markers [glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), Bcl-2-associated X (Bax) and cleaved caspase-3]. The GFP-RFP-LC3 adenovirus was used to assay the change of autophagic flux. Our results showed that berberine could increase the viability of PC12 cells, decrease the concentrations of ROS after OGD/R treatment, and suppress OGD/R-induced ER stress and autophagy. Moreover, the results revealed the involvement of the mammalian target of rapamycin (mTOR) pathway in the induction of autophagy, and berberine could activate the phosphorylation of mTOR and thus mitigate autophagy. In conclusion, our study suggested that berberine may protect against OGD/R-induced apoptosis by regulating ER stress and autophagy, and it holds promises in the treatment of cerebral I/R injury.

Astrocytic α7 Nicotinic Receptor Activation Inhibits Amyloid-ß Aggregation by Upregulating Endogenous αB-crystallin through the PI3K/Akt Signaling Pathway.

BACKGROUND: ß-amyloid (Aß) aggregation plays an important role in the pathogenesis of Alzheimer's disease (AD), and astrocytes can significantly inhibit Aß aggregation. Astrocytic α7 Neuronal Nicotinic Acetylcholine Receptor (nAChR) upregulation detected in the AD brains is closely associated with Aß deposits. However, the relationships between the astrocytic α7 nAChRs and Aß aggregation remain unclear. METHODS: The Aß oligomers levels in astrocytic cell lysates and culture medium were measured after treatment with nicotine or co-treatment with a Phosphatidylinositol 3-Kinase (PI3K)-protein kinase B (Akt) inhibitor. The level of αB-Crystallin (Cryab) in astrocytes treated with nicotine for different times or co-treated with α7 nAChR antagonists as well as co-incubated with a PI3K or mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor was determined by western blotting. RESULTS: In this study, nicotine pre-treatment in primary astrocytes markedly inhibited Aß aggregation and upregulated endogenous astrocytic Cryab, while the nicotine-mediated neuroprotective effect was reversed by pre-treatment with a selective α7 nAChR antagonist. Furthermore, this neuroprotection against Aß aggregation was suppressed by LY294002, a PI3K inhibitor. Pre-treatment with nicotine significantly increased the levels of phosphorylated Akt, an effector of PI3K in astrocytes. CONCLUSION: α7 nAChR activation and PI3K/Akt signaling transduction contributed to nicotinemediated neuroprotection against Aß aggregation by modulating endogenous astrocytic Cryab.