Mechanisms of dihydromyricetin against hepatocellular carcinoma elucidated by network pharmacology combined with experimental validation.

CONTEXT: Dihydromyricetin (DMY) is extracted from vine tea, a traditional Chinese herbal medicine with anti-cancer, liver protection, and cholesterol-lowering effects. OBJECTIVE: This study investigated the mechanism of DMY against hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Potential DMY, HCC, and cholesterol targets were collected from relevant databases. PPI networks were created by STRING. Then, the hub genes of co-targets, screened using CytoHubba. GO and KEGG pathway enrichment, were performed by Metascape. Based on the above results, a series of in vitro experiments were conducted by using 40-160 µM DMY for 24 h, including transwell migration/invasion assay, western blotting, and Bodipy stain assay. RESULTS: Network pharmacology identified 98 common targets and 10 hub genes of DMY, HCC, and cholesterol, and revealed that the anti-HCC effect of DMY may be related to the positive regulation of lipid rafts. Further experiments confirmed that DMY inhibits the proliferation, migration, and invasion of HCC cells and reduces their cholesterol levels in vitro. The IC50 is 894.4, 814.4, 467.8, 1,878.8, 151.8, and 156.9 µM for 97H, Hep3B, Sk-Hep1, SMMC-7721, HepG2, and Huh7 cells, respectively. In addition, DMY downregulates the expression of lipid raft markers (CAV1, FLOT1), as well as EGFR, PI3K, Akt, STAT3, and Erk. DISCUSSION AND CONCLUSION: The present study reveals that DMY suppresses EGFR and its downstream pathways by reducing cholesterol to disrupt lipid rafts, thereby inhibiting HCC, which provides a promising candidate drug with low toxicity for the treatment of HCC.

Characteristics and pathogenesis of chemokines in the post-stroke stage.

Chemokines, as small molecular proteins, play a crucial role in the immune and inflammatory responses after stroke. A large amount of evidence showed chemokines and their receptors were increasingly recognized as potential targets for stroke treatment, which were involved in the processing of neovascularization, neurogenesis, and neural network reconstruction. In this review, we summarized the characteristics of chemokine alterations throughout the post-stroke nerve repair phase to gain insight into the pathological mechanisms of chemokines and find effective therapeutic targets for stroke.

Novel antidepressant mechanism of ginsenoside Rg1: Regulating biosynthesis and degradation of connexin43.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C. A. Meyer is a valuable medicinal herb and "alternative" remedy for the prevention and treatment of depression. Dysfunction of connexin43 (Cx43)-gap junction in astrocytes is predisposed to the precipitation of depression. Ginsenoside Rg1 (Rg1), the main bioactive constituent extracted from ginseng, is efficacious in the management of depression by upregulating the content of Cx43. Our previous results indicated that pretreatment with Rg1 significantly improved Cx43-gap junction in corticosterone (CORT)-treated astrocytes. However, the antidepressant mechanism underlying how Rg1 upregulates Cx43-gap junction in astrocytes hasn't been proposed. AIM OF THE STUDY: To dissect the mechanisms of Rg1 controlling Cx43 levels in primary astrocytes. METHODS: We examined the changes of the level of Cx43 mRNA, the degradation of Cx43, as well as the ubiquitin-proteasomal and autophagy-lysosomal degradation pathways of Cx43 followed by Rg1 prior to CORT in rat primary astrocytes isolated from prefrontal cortex and hippocampus. Furthermore, the recognized method of scrape loading/dye transfer was performed to detect Cx43-gap junctional function, an essencial indicator of the antidepressant effect. RESULTS: Pretreatment with Rg1 could reverse CORT-induced downregulation of Cx43 biosynthesis, acceleration of Cx43 degradation, and upregulation of two Cx43 degradation pathways in primary astrocytes. CONCLUSION: The findings in the present study provide the first evidence highlighting that Rg1 increases Cx43 protein levels through the upregulation of Cx43 mRNA and downregulation of Cx43 degradation, which may be attributed to the effect of Rg1 on the ubiquitin-proteasomal and autophagy-lysosomal degradation pathways of Cx43.

Kanglaite enhances the efficacy of cisplatin in suppression of hepatocellular carcinoma via inhibiting CKLF1 mediated NF-κB pathway and regulating transporter mediated drug efflux.

ETHNOPHARMACOLOGICAL RELEVANCE: Kanglaite (KLT) is an active extract of the Coix lacryma-jobi seed, which can benefit Qi and nourish Yin, and disperse the accumulation of evils. It is used as a biphasic broad-spectrum anti-cancer drug, and shows synergistic effects with radiotherapy and chemotherapy. However, the mechanism of KLT combined with cisplatin (CDDP) against hepatocellular carcinoma (HCC) has not been elucidated. AIM OF THE STUDY: The aim of present study was to investigate the potential synergistic effects of KLT and CDDP on HepG2 cells, discussing the possible mechanisms from the perspective of CKLF1 and NF-κB mediated inflammatory response and chemoresistance, and the involvement of drug efflux transporters. MATERIALS AND METHODS: CDDP injured HepG2 cells were used to investigate the effects of KLT on chemotherapeutics treated HCC. Effects of KLT pretreatment on CDDP injured HepG2 cells were determined by MTT, wound healing assay, and transwell assay. Expression of chemokine-like factor 1 (CKLF1) and activation of nuclear factor κB (NF-κB) were examined by qPCR, western blot, and immunofluorescence staining. Furthermore, to study the role of CKLF1 in KLT mediated effects on this CDDP injured HCC cell model, HepG2 cells overexpressed with CKLF1 gene were used. Cell viability and NF-κB activation were investigated. Moreover, TNF-α and IL-1ß levels were measured by Elisa analysis and western blot to evaluate the inflammatory response. Additionally, ATP-binding cassette (ABC) drug efflux transporters, MDR1, MRP2, and BCRP were also determined in present study. RESULTS: KLT pretreatment followed by CDDP treatment was found to show synergistic effects, which showed by decreased cell viability, migration and invasion ability of HepG2 cells. Expression of CKLF1 enhanced significantly in CDDP treated HepG2 cells, and KLT decreased this elevation obviously. Furthermore, CDDP activated NF-κΒ and promoted translocation of NF-κB toward the nucleus. KLT inhibited the activation of NF-κΒ, which sensitized cancer cells. Overexpression of CKLF1 reversed the effects of KLT on CDDP injured HepG2 cells, which exhibited by increased cell viability and enhanced activation of NF-κΒ. CDDP induced NF-κΒ activation could also lead to excessive inflammatory response, and KLT can suppress the aggravating inflammation which may be beneficial for tumor progression. Furthermore, we found that ABC drug efflux transporters MDR1, MRP2, and BCRP in CDDP treated HepG2 cells were decreased when pretreated with KLT. CONCLUSIONS: KLT pretreatment may increase the effects of CDDP on HepG2 cells, by exhibiting cooperative effects on suppression of HepG2 cells. The mechanisms may partly by inhibiting CKLF1 mediated NF-κB pathway, which may contribute to inflammation of tumor microenvironment and chemoresistance of CDDP. Inhibition of transporter-mediated drug efflux is also involved in KLT mediated sensitization effects of CDDP.

CKLF1/CCR5 axis is involved in neutrophils migration of rats with transient cerebral ischemia.

BACKGROUND: Chemokine-like factor 1 (CKLF1) is a chemokine increased significantly in ischemic brain poststroke. It shows chemotaxis effects on various immune cells, but the mechanisms of CKLF1 migrating neutrophils are poorly understood. Recent studies have provided evidence that CC chemokine receptor 5 (CCR5), a receptor of CKLF1, is involved in ischemic stroke. PURPOSES: To investigate the effects of HIF-1α guided AAV in ischemic brain, investigating the outcome of stroke, and examining the involvement of CKLF1/CCR5 axis in recruitment of neutrophils. RESULTS: HIF-1α guided AAV knocked down CKLF1 in ischemic area and alleviated brain damage of rats. CKLF1 migrated neutrophils through CCR5, worsening inflammatory responses. Akt/GSK-3ß pathway may involve in CKLF1/CCR5 axis guided neutrophils chemotaxis. CONCLUSIONS: CKLF1/CCR5 axis is involved in neutrophils migration of rats with transient cerebral ischemia. CKLF1/CCR5 axis may be a useful target for stroke therapy.

CKLF1 Aggravates Focal Cerebral Ischemia Injury at Early Stage Partly by Modulating Microglia/Macrophage Toward M1 Polarization Through CCR4.

CKLF1 is a chemokine with increased expression in ischemic brain, and targeting CKLF1 has shown therapeutic effects in cerebral ischemia model. Microglia/macrophage polarization is a mechanism involved in poststroke injury expansion. Considering the quick and obvious response of CKLF1 and expeditious evolution of stroke lesions, we focused on the effects of CKLF1 on microglial/macrophage polarization at early stage of ischemic stroke (IS). The present study is to investigate the CKLF1-mediated expression of microglia/macrophage phenotypes in vitro and in vivo, discussing the involved pathway. Primary microglia culture was used in vitro, and mice transient middle cerebral artery occlusion (MCAO) model was adopted to mimic IS. CKLF1 was added to the primary microglia for 24 h, and we found that CKLF1 modulated primary microglia skew toward M1 phenotype. In mice transient IS model, CKLF1 was stereotactically microinjected to the lateral ventricle of ischemic hemisphere. CKLF1 aggravated ischemic injury, accompanied by promoting microglia/macrophage toward M1 phenotypic polarization. Increased expression of pro-inflammatory cytokines and decreased expression of anti-inflammatory cytokines were observed in mice subjected to cerebral ischemia and administrated with CKLF1. CKLF1-/- mice were used to confirm the effects of CKLF1. CKLF1-/- mice showed lighter cerebral damage and decreased M1 phenotype of microglia/macrophage compared with the WT control subjected to cerebral ischemia. Moreover, NF-κB activation enhancement was detected in CKLF1 treatment group. Our results demonstrated that CKLF1 is an important mediator that skewing microglia/macrophage toward M1 phenotype at early stage of cerebral ischemic injury, which further deteriorates followed inflammatory response, contributing to early expansion of cerebral ischemia injury. Targeting CKLF1 may be a novel way for IS therapy.

Ginsenoside Rg1 prevents acetaminophen-induced oxidative stress and apoptosis via Nrf2/ARE signaling pathway.

Inappropriate use of acetaminophen (APAP) can lead to morbidity and mortality secondary to hepatic necrosis. Ginsenoside Rg1 is a major active ingredient in processed Panax ginseng, which is proved to elicit biological effects. We hypothesized the beneficial effect of Rg1 on APAP-mediated hepatotoxicity was through Nrf2/ARE pathway. The study was conducted in cells and mice, comparing the actions of Rg1. Rg1 significantly improved cell survival rates and promoted the expression of antioxidant proteins. Meanwhile, Rg1 reduced the excessive ROS and the occurrence of cell apoptosis, which were related to Nrf2/ARE pathway. Expression of Nrf2 has a certain cell specificity.

CZ-7, a new derivative of Claulansine F, ameliorates 2VO-induced vascular dementia in rats through a Nrf2-mediated antioxidant responses.

Vascular dementia (VD) results from accumulated damage in the vascular system, which is characterized by progressive impairments in memory and cognition and is second only to Alzheimer's disease (AD) in prevalence among all types of dementia. In contrast to AD, there is no FDA-approved treatment for VD owing to its multiple etiologies. In this study, we investigated whether CZ-7, a new derivative of Claulansine F (Clau F) with verified neuroprotective activity in vitro, could ameliorate the cognitive impairment of rats with permanent occlusion of bilateral common carotid arteries (2VO) and its potential mechanisms of action. The 2VO rats were orally administered CZ-7 (10, 20, 40 mg/kg) from day 27 to day 53 post-surgery. Morris water maze tests conducted at day 48-51 revealed that CZ-7 administration significantly reduced the escape latency in 2VO rats. After the rats were sacrificed on day 53, morphological studies using Nissl and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining showed that administration of CZ-7 markedly attenuated the pathological changes in CA1-CA3 area of the hippocampus, including neuronal cell loss, nuclear shrinkage, and dark staining of neurons, and significantly decreased the chronic cerebral hypoperfusion-induced cell loss. Klüver-Barrera staining study revealed that CZ-7 administration significantly improved the white matter lesions. 8-OHdG and reactive oxygen species (ROS) immunofluorescent analyses showed that CZ-7 administration significantly decreased oxidative stress in CA1-CA3 area of the hippocampus. Finally, we found that the CZ-7-improved oxidative stress might be mediated via the Nrf2 pathway, evidenced by the double immunofluorescent staining of Nrf2 and the elevation of expression levels of oxidative stress proteins HO-1 and NQO1. In conclusion, CZ-7 has therapeutic potential for VD by alleviating oxidative stress injury through Nrf2-mediated antioxidant responses.

NK cells in cerebral ischemia.

As a vital cell type in immune system and infiltrating cells in ischemic brain, NK cells can bridge the crosstalk between immune system and nervous system in stroke setting. The mechanism of action of NK cells is complicated, involving direct and indirect actions. NK cells are closely associated with poststroke inflammation, immunodepression and infections. The excessive inflammatory response in ischemic brain is one of the important causes for aggravating cerebral ischemic injury. Besides the inflammation induced by ischemia itself, thrombolytic drug tissue plasminogen activator (tPA) administration could also induce deteriorative inflammation, which is unfavorable for stroke control and recovery. Regulating NK cells may has the potential to modulate the immune response, limiting the development of ischemic damage and getting better outcome. In addition, post-stroke immunosuppression may lead to infections which contribute to higher severity and mortality of ischemic stroke (IS). Targeting NK cells may help to find novel pathways for IS therapy, which can both ameliorate the infarction itself, but also reduce the infectious complications. NK cells may also link IS and related diseases, suggesting NK cells can be used as a diagnostic or prognostic biomarker for IS prevention and treatment.

Ginsenoside Rg1 protects against ischemic/reperfusion-induced neuronal injury through miR-144/Nrf2/ARE pathway.

Ginsenoside Rg1 (Rg1), a saponin extracted from Panax ginseng, has been well documented to be effective against ischemic/reperfusion (I/R) neuronal injury. However, the underlying mechanisms remain obscure. In the present study, we investigated the roles of Nrf2 and miR-144 in the protective effects of Rg1 against I/R-induced neuronal injury. In OGD/R-treated PC12 cells, Rg1 (0.01-1 µmol/L) dose-dependently attenuated the cell injury accompanied by prolonging nuclear accumulation of Nrf2, enhancing the transcriptional activity of Nrf2, as well as promoting the expression of ARE-target genes. The activation of the Nrf2/ARE pathway by Rg1 was independent of disassociation with Keap1, but resulted from post-translational regulations. Knockdown of Nrf2 abolished all the protective changes of Rg1 in OGD/R-treated PC12 cells. Furthermore, Rg1 treatment significantly decreased the expression of miR-144, which downregulated Nrf2 production by targeting its 3'-untranlated region after OGD/R. Knockdown of Nrf2 had no effect on the expression of miR-144, suggesting that miR-144 was an upstream regulator of Nrf2. We revealed that there was a direct binding between Nrf2 and miR-144 in PC12 cells. Application of anti-miR-144 occluded the activation of the Nrf2/ARE pathway by Rg1 in OGD/R-treated PC12 cells. In tMCAO rats, administration of Rg1 (20 mg/kg) significantly alleviated ischemic injury, and activated Nrf2/ARE pathway. The protective effects of Rg1 were abolished by injecting of AAV-HIF-miR-144-shRNA into the predicted ischemic penumbra. In conclusion, our results demonstrate that Rg1 alleviates oxidative stress after I/R through inhibiting miR-144 activity and subsequently promoting the Nrf2/ARE pathway at the post-translational level.

Ursodeoxycholic acid protects interstitial Cajal-like cells in the gallbladder from undergoing apoptosis by inhibiting TNF-α expression.

Hypomotility is a common symptom of gallstone disease, which is accompanied by a loss of interstitial Cajal-like cells (ICLCs) in the gallbladder. Ursodeoxycholic acid (UDCA) is widely used in treating gallstone disease, and has shown anti-apoptotic and anti-inflammatory effects apart from its ability to dissolve gallstones. In this study, we investigated the anti-apoptotic and anti-inflammatory effects of UDCA on ICLCs in guinea pigs with gallstones. Guinea pigs were fed a high-cholesterol diet for 8 weeks to induce the formation of gallstones. A group of animals was administered UDCA (50 mg·kg-1·d-1, ig) simultaneously. At the end of 8 weeks, the animals were euthanized with anesthesia, cholecystectomy was performed immediately and gallbladder was collected for further analysis. We showed that in the model group the contractility of gallbladder muscle strips in response to both acetylcholine (ACh) and CCK-8 was severely impaired, which was significantly improved by UDCA administration. Furthermore, UDCA administration significantly reduced the apoptotic ratio of ICLCs, based on the observation of co-localization imaging of apoptotic cells and c-kit-positive cells. Western blotting analysis and real-time PCR results revealed that the TNF-α/Caspase8/Caspase3 pathway was suppressed in the UDCA-treated animals, confirming the anti-apoptotic effect of UDCA in the gallbladder. The H&E staining showed that UDCA administration significantly attenuated inflammatory cell infiltration in the gallbladder wall. In conclusion, UDCA can protect ICLCs in the gallbladder from undergoing apoptosis by inhibiting the TNF-α/Caspase8/caspase3 pathway.

Progress in pharmacological research of chemokine like factor 1 (CKLF1).

Currently, the research of chemokines has penetrated into many fields of life science. A new kind of chemokines, chemokine like factor 1 (CKLF1), which is cloned through suppression subtractive hybridisation (SSH) technology is expressed widely in human body, especially in the lung and peripheral blood leukocytes. CKLF1 has a broad spectrum of chemotaxic activity for many cells, such as lymphocytes, macrophages, bone marrow cells, nerve cells and so on. In addition, CKLF1 also stimulates the regeneration of skeletal muscle cells in vivo. Collecting data derived from our and other laboratories show that CKLF1 has an important relationship with allergic diseases, autoimmune diseases, tumors, cardio-cerebrovascular diseases and so on. Therefore, there be an important theoretical purport and applied value to make a summary of pharmacological progress of CKLF1.

RTP801 is a critical factor in the neurodegeneration process of A53T α-synuclein in a mouse model of Parkinson's disease under chronic restraint stress.

BACKGROUND AND PURPOSE: Recently, the incidence of Parkinson's disease has shown a tendency to move to a younger population, linked to the constantly increasing stressors of modern society. However, this relationship remains obscure. Here, we have investigated the contribution of stress and the mechanisms underlying this change. EXPERIMENTAL APPROACH: Ten-month-old α-synuclein A53T mice, a model of Parkinson's disease (PD), were treated with chronic restraint stress (CRS) to simulate a PD-sensitive person with constant stress stimulation. PD-like behavioural tests and pathological changes were evaluated. Differentiated PC12-A53T cells were treated with corticosterone in vitro. We used Western blot, microRNA expression analysis, immunofluorescence staining, dual luciferase reporter assay and HPLC electrochemical detection to assess cellular and molecular networks after stress treatment. In vivo, stereotaxic injection of shRNA lentivirus was used to confirm our in vitro results. KEY RESULTS: The protein RTP801 is encoded by DNA-damage-inducible transcript 4, and it was specifically increased in dopaminergic neurons of the substantia nigra after CRS treatment. RTP801 was post-transcriptionally inhibited by the down-regulation of miR-7. Delayed turnover of RTP801, through the inhibition of proteasome degradation also contributed to its high content. Elevated RTP801 blocked autophagy, thus increasing accumulation of oligomeric α-synuclein and aggravating endoplasmic reticulum stress. RTP801 inhibition alleviated the symptoms of neurodegeneration during this process. CONCLUSIONS AND IMPLICATIONS: RTP801 is a promising target for the treatment of PD, especially for PD-sensitive patients who live under increased social pressure. Down-regulation of RTP801 could inhibit the current tendency to an earlier onset of PD.

Autophagy: An Exposing Therapeutic Target in Atherosclerosis.

Autophagy is an evolutionarily conserved catabolic process whereby the cytoplasmic contents of a cell are sequestered within autophagosomes through a lysosome-dependent pathway. Increasing evidence shows that this process is of great importance in a wide range of diseases, including atherosclerosis (AS). Autophagy can be modulated in advanced AS plaques by cytokines, reactive lipids, lipopolysaccharides, advanced glycation end products, and microRNAs. Autophagy exerts both protective and detrimental functions in vascular disorders. However, despite an increasing interest in autophagy, it remains an underestimated and overlooked phenomenon in AS. Therefore, the precise role of autophagy and its relationship with apoptosis need to be described. This review highlights recent findings on the autophagy activities and signaling pathways in endothelial cells, macrophages, and smooth muscle cells that are accompanied by apoptosis in AS. We conclude with recent studies on autophagy modulation as a new therapeutic approach to treat AS.