The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis.

Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.

Define Mesenchymal Stem Cell from Its Fate: Biodisposition of Human Mesenchymal Stem Cells in Normal and Concanavalin A-Induced Liver Injury Mice.

The pharmaceutical industry and clinical trials have been revolutionized mesenchymal stem cell-based therapeutics. However, the pharmacokinetics of transplanted cells has been little characterized in their target tissues under healthy or disease condition. A quantitative polymerase chain reaction analytical method with matrix effect was developed to track the biodistribution of human mesenchymal stem cells in normal mice and those with Concanavalin A (Con A)-induced liver injury. Mesenchymal stem/stromal cell (MSC) disposition in blood and different organs were compared, and relevant pharmacokinetic parameters were calculated. Human MSCs (hMSCs) and mouse MSCs (mMSCs) displayed a very similar pharmacokinetic profile in all tested doses: about 95% of the detected hMSCs accumulated in the lung and 3% in the liver, and almost negligible cells were detected in other tissues. A significant double peak of hMSC concentration emerged in the lung within 1-2 hours after intravenous injection, as with mMSCs. Prazosin, a vasodilator, could eliminate the second peak in the lung and increase its Cmax and area under the concentration-time curve (AUC) by 10% in the first 2 hours. The injury caused by Con A was significantly reduced by hMSCs, and the Cmax and AUC0-8 (AUC from time 0 to 8 hours) of cells in the injured liver decreased by 54 and 50%, respectively. The Cmax and AUC would be improved with the alleviation of congestion through the administration of heparin. The study provides a novel insight into the pharmacokinetics of exogenous MSCs in normal and Con A-induced liver injury mice, which provides a framework for optimizing cell transplantation. SIGNIFICANCE STATEMENT: Mesenchymal stem/stromal cells (MSCs) are known for their potential as regenerative therapies in treating several diseases, but an insufficient understanding of the pharmacokinetics of MSCs restricts their future application. The current study was the first to elucidate the pharmacokinetics and possible factors, including dosage, species, and derived sources, in a systematic way. The study further revealed that Concanavalin A-induced liver injury significantly prevented cells from entering the injury site, which could be reversed by the diminished congestion achieved by heparin.

Anti-Fibrosis Effects of Magnesium Lithospermate B in Experimental Pulmonary Fibrosis: By Inhibiting TGF-ßRI/Smad Signaling.

Pulmonary fibrosis is a severe and irreversible interstitial pulmonary disease with high mortality and few treatments. Magnesium lithospermate B (MLB) is a hydrosoluble component of Salvia miltiorrhiza and has been reported to have antifibrotic effects in other forms of tissue fibrosis. In this research, we studied the effects of MLB on pulmonary fibrosis and the underlying mechanisms. Our results indicated that MLB treatment (50 mg/kg) for seven days could attenuate bleomycin (BLM)-induced pulmonary fibrosis by reducing the alveolar structure disruption and collagen deposition in the C57 mouse model. MLB was also found to inhibit transforming growth factor-beta (TGF-ß)-stimulated myofibroblastic transdifferentiation of human lung fibroblast cell line (MRC-5) cells and collagen production by human type II alveolar epithelial cell line (A549) cells, mainly by decreasing the expression of TGF-ß receptor I (TGF-ßRI) and regulating the TGF-ß/Smad pathway. Further studies confirmed that the molecular mechanisms of MLB in BLM-induced pulmonary fibrosis mice were similar to those observed in vitro. In summary, our results demonstrated that MLB could alleviate experimental pulmonary fibrosis both in vivo and in vitro, suggesting that MLB has great potential for pulmonary fibrosis treatment.

A novel bile acid analog, A17, ameliorated non-alcoholic steatohepatitis in high-fat diet-fed hamsters.

Being endocrine signaling molecules that regulate lipid metabolism and affect energy balance, bile acids are potential drug candidates for non-alcoholic steatohepatitis (NASH). Obeticholic acid (OCA) could improve NASH accompanied by significant side effects. Therefore, it is worthwhile to develop safer and more effective bile acid analogs. In this study, a new bile acid analog A17 was synthesized and its potential anti-NASH effects were assessed in vitro and in vivo. The impact of A17 on steatosis was investigated in the rat primary hepatocytes challenged with oleic acid. It was found that A17 alleviated lipid accumulation by reducing fatty acid (FA) uptake and promoting FA oxidation. The reduction of FA uptake came from inhibiting fatty acid translocase (Cd36) expression. The promotion of FA oxidation came from stimulating the phosphorylation of adenosine monophosphate (AMP)-activated protein kinase alpha (AMPKα). In addition, A17 reduced lipopolysaccharide-induced inflammation in Raw264.7 cells by activating Takeda G protein-coupled receptor 5 (TGR5). In in vivo study, male Golden Syrian hamsters were fed with high fat (HF) diet and then treated with 50 mg/kg/d A17 for 6 weeks. A17 lowered the lipid profiles and liver enzyme levels in serum and improved liver pathological conditions with less side effects compared with OCA. Further studies confirmed that the molecular mechanisms of A17 in vivo were similar to those in vitro. In conclusion, a novel bile acid analog A17 was identified to ameliorate NASH in HF-fed hamsters. The potential mechanisms could be contributed to reducing FA uptake, stimulating FA oxidation and relieving inflammation.

Reprogramming Tumor-Associated Macrophages To Reverse EGFRT790M Resistance by Dual-Targeting Codelivery of Gefitinib/Vorinostat.

Gefitinib is a first-line therapy in the EGFR-mutated nonsmall cell lung cancer (NSCLC). However, the development of drug resistance is almost unavoidable, thus leading to an unsustainable regimen. EGFRT790M mutation is the major cause responsible for the molecular-targeting therapy failure in NSCLC. Although the recently approved osimertinib is effective for the EGFRT790M-positive NSCLC, the osimertinib-resistant EGFR mutation is rapidly developed, too. In this study, we proposed a tumor-associated macrophage (TAM) reprogramming strategy for overcoming the EGFRT790M-associated drug resistance via a dual-targeting codelivery system of gefitinib/vorinostat that acted on both TAM with overexpression of mannose receptors and the HER-2 positive NSCLC cells. The trastuzumab-modified, mannosylated liposomal system was able to repolarize the protumor M2 phenotype to the antitumor M1 and cause the elevating ROS in the cancer cells, consequently modulating the intracellular redox balance via ROS/NOX3/MsrA axis. The suppressed MsrA facilitated the EGFRT790M degradation through 790M oxidation by ROS, thus resensitizing the EGFRT790M-positive cells to gefitinib. The dual-targeting codelivery and TAM-reprogramming strategies provided a potential method for rescuing the EGFRT790M-caused resistance to tyrosine kinase inhibitor treatment.

Codelivery of dihydroartemisinin and doxorubicin in mannosylated liposomes for drug-resistant colon cancer therapy.

Multidrug resistance (MDR) is a major hurdle in cancer chemotherapy and makes the treatment benefits unsustainable. Combination therapy is a commonly used method for overcoming MDR. In this study we investigated the anti-MDR effect of dihydroartemisinin (DHA), a derivative of artemisinin, in combination with doxorubicin (Dox) in drug-resistant human colon tumor HCT8/ADR cells. We developed a tumor-targeting codelivery system, in which the two drugs were co-encapsulated into the mannosylated liposomes (Man-liposomes). The Man-liposomes had a mean diameter of 158.8 nm and zeta potential of -15.8 mV. In the HCT8/ADR cells that overexpress the mannose receptors, the Man-liposomes altered the intracellular distribution of Dox, resulting in a high accumulation of Dox in the nuclei and thus displaying the highest cytotoxicity (IC50=0.073 µg/mL) among all the groups. In a subcutaneous HCT8/ADR tumor xenograft model, administration of the Man-liposomes resulted in a tumor inhibition rate of 88.59%, compared to that of 47.46% or 70.54%, respectively, for the treatment with free Dox or free Dox+DHA. The mechanisms underlying the anti-MDR effect of the Man-liposomes involved preferential nuclear accumulation of the therapeutic agents, enhanced cancer cell apoptosis, downregulation of Bcl-xl, and the induction of autophagy.

Cell-penetrating apoptotic peptide/p53 DNA nanocomplex as adjuvant therapy for drug-resistant breast cancer.

Drug resistance becomes a formidable challenge against effective cancer therapy. Defective apoptosis in cancer cells is a key factor responsible for chemoresistance or radioresistance. Promoting apoptosis is an important method to sensitize the resistant cells, thereby achieving successful treatment for MDR cancer. We present a strategy of codelivery of apoptotic AVPI peptide and p53 DNA as apoptosis-induction adjuvant therapy for combating the resistant breast cancer. AVPI tetrapeptide is poorly cell-permeable, thereby with very limited value for therapeutic use. Cell-penetrating chimeric AVPI derivative was developed by modification with an octa-arginine sequence (R8). The AVPIR8 is able to not only efficiently penetrate into tumor cells but also work as a vector for gene delivery by forming nanocomplexes based on its cationic R8 moiety. The combination of AVPIR8/p53 DNA was selected for targeting apoptotic pathways, thereby sensitizing the cancer cells to chemotherapeutics. The anti-MDR effect was demonstrated both in vitro and in vivo. The synergistic use of AVPIR8/p53 significantly increased the sensitivity of the resistant tumor cells to the cytotoxic agent doxorubicin by inducing apoptosis, as demonstrated in the cellular studies. Importantly, the treatment improvement was also observed in the animal studies with resistant breast tumor model. Coadministration of AVPIR8/p53 enabled a full arrest of tumor growth combined with a reduced DOX dose, yielding a productive and safe cancer treatment.

Gigantol ameliorates CCl4-induced liver injury via preventing activation of JNK/cPLA2/12-LOX inflammatory pathway.

Arachidonic acid (AA) signaling pathway is an important constituent of inflammatory processes. In our previous study, it was found that dihydro-stilbene gigantol relieved hepatic inflammation in mice with CCl4-induced acute liver injury. This study aimed to investigate the involvement of arachidonate metabolic cascade in this process. Our results showed CCl4 activated AA metabolism with the evidence of cPLA2 phosphorylation, which was dependent on the MAPK/JNK activation. Pretreatment with JNK inhibitor SU3327 or gigantol abolished the cPLA2 activation, along with the attenuation of liver damage. Besides, gigantol markedly decreased immune cells activation. Metabolomic analysis revealed that gigantol universally reversed the upregulation of major AA metabolites in injured mouse livers induced by CCl4, especially 12-hydroxyeicosatetraenoic acid (12-HETE). Gigantol also decreased the mRNA and protein expression of platelet-, and leukocyte-type 12-lipoxxygenase (LOX) in the liver. Furthermore, pan-LOX inhibitor nordihydroguaiaretic acid (NDGA) and specific 12-LOX inhibitors baicalein and ML351 attenuated the liver injury to the same extent as gigantol. Overall, our study elucidated a comprehensive profile of AA metabolites during hepatic inflammation caused by CCl4, highlighting the role of 12-LOX-12-HETE pathway in this process. And gigantol alleviated liver inflammation partly through inhibiting the JNK/cPLA2/12-LOX pathway.

The Protective Effect of Magnesium Lithospermate B on Hepatic Ischemia/Reperfusion via Inhibiting the Jak2/Stat3 Signaling Pathway.

Acute inflammation is an important component of the pathogenesis of hepatic ischemia/reperfusion injury (HIRI). Magnesium lithospermate B (MLB) has strong neuroprotective and cardioprotective effects. The purpose of this study was to determine whether MLB had underlying protective effects against hepatic I/R injury and to reveal the potential mechanisms related to the hepatoprotective effects. In this study, we first examined the protective effect of MLB on HIRI in mice that underwent 1 h ischemia followed by 6 h reperfusion. MLB pretreatment alleviated the abnormal liver function and hepatocyte damage induced by I/R injury. We found that serum inflammatory cytokines, including IL-6, IL-1ß, and TNF-α, were significantly decreased by MLB during hepatic ischemia/reperfusion (I/R) injury, suggesting that MLB may alleviate hepatic I/R injury via inhibiting inflammatory signaling pathways. Second, we investigated the protein level of p-Jak2/Jak2 and p-Stat3/Stat3 using Western blotting and found that MLB could significantly inhibit the activation of the Jak2/Stat3 signaling pathway, which was further verified by AG490 in a mouse model. Finally, the effect of MLB on the Jak2/Stat3 pathway was further assessed in an in vitro model of RAW 264.7 cells; 1 µg/ml LPS induced the secretion of inflammatory mediators, including IL-6, TNF-α, and activation of the Jak2/Stat3 signaling pathway. MLB significantly inhibited the abnormal secretion of inflammatory factors and the activation of the Jak2/Stat3 signaling pathway in RAW264.7 cells. In conclusion, MLB was found for the first time to reduce inflammation induced by hepatic I/R via suppressing the Jak2/Stat3 pathway.

Exploration of the hepatoprotective chemical base of an orally administered herbal formulation (YCHT) in normal and CCl4-intoxicated liver injury rats. Part 2: Hepatic disposition in vivo and hepatoprotective activity in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Yin-Chen-Hao Tang (YCHT) has been a very popular, hepatoprotective three-herb formula with an unclear chemical base. AIM OF THIS STUDY: To reveal the hepatoprotective chemical base of oral-dosed YCHT, we bridged the hepatic disposition of six compounds in vivo and their hepatoprotection in vitro. MATERIALS AND METHODS: In vivo, following the oral administration of YCHT in normal and CCl4-induced liver injury rats, the determinations of chlorogenic acid, 4-hydroxyacetophenone, geniposide, genipin, rhein and emodin were conducted in the portal vein plasma, the liver, and the systemic plasma. In vitro, the hepatoprotective activities of these compounds were determined in the CCl4-induced HepG2 cells. RESULTS: Consistent with the highest content in YCHT, geniposide had the highest exposure in vivo. Inconsistent with the negligible content, rhein, 4-hydroxyacetophenone, emodin and genipin showed substantial hepatic accumulations. In contrast, chlorogenic acid, an ingredient that has a high content in YCHT, elicited no hepatic exposure. In normal rats, the hepatic disposition prevented the compounds entering into the systemic plasma from the portal vein plasma by 44.9-100%, except for rhein. CCl4-induced liver injury caused a decreased hepatic exposure of 4-hydroxyacetophenone, rhein and emodin by 50%. In vitro, all six compounds exerted the hepatoprotection by increasing cell viability, decreasing hepatic marker enzymes and inhibiting lipid peroxidation at varying levels. CONCLUSION: Geniposide, rhein, emodin, 4-hydroxyacetophenone and genipin directly resisted liver injury in oral-dosed YCHT, while chlorogenic acid likely played an indirect role. This study proved that YCHT exerted hepatoprotection through multiple components and multiple actions. However, close attention should be paid to the possible side effects and oral dosage of YCHT in clinics.

Poly-γ-glutamic acid-based GGT-targeting and surface camouflage strategy for improving cervical cancer gene therapy.

Polycation-based delivery presents a major method for non-viral gene therapy. However, the disadvantages of cationic vectors are their tendencies to be captured and eliminated by the reticuloendothelial system due to their excessive positive charges and nonspecific interaction with normal cells that leads to adverse effects. PEGylation was applied to solve these major problems. Yet, PEG chains can severely compromise cellular uptake and yield unsatisfying efficiency resulting in a so-called PEG dilemma. We developed a γ-PGA-based GGT-targeting and surface camouflage strategy by constructing a ternary complex system via a layer-by-layer self-assembly method. The biodegradable polyanion γ-PGA could protect the PEI/pDNA complexes from interaction with the body fluid components; however, in endosome, the polyanion facilitated the intracellular release of PEI/pDNA. The γ-PGA/PEI/pDNA nanoparticles possessed a markedly improved serum-tolerant capability. More importantly, γ-PGA interacts with the tumor-associated γ-glutamyl transpeptidase (GGT) that can mediate endocytosis of the nanoparticles. With pTRAIL as the therapeutic gene, the γ-PGA/PEI/pTRAIL nanoparticles effectively inhibited tumor cell proliferation by inducing cell apoptosis and arresting cell cycles. The in vivo results displayed effective suppression of tumor growth, and high treatment efficacy in the mice bearing cervical tumor. The γ-PGA-based GGT-targeting and surface camouflage strategy is a potential method for improved gene delivery and cancer therapy.

Recent progress in microRNA delivery for cancer therapy by non-viral synthetic vectors.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Because of significant changes in their expression in cancer, miRNAs are believed to be key factors in cancer genetics and to have potential as anticancer drugs. However, the delivery of miRNAs is limited by many barriers, such as low cellular uptake, immunogenicity, renal clearance, degradation by nucleases, elimination by phagocytic immune cells, poor endosomal release, and untoward side effects. Nonviral delivery systems have been developed to overcome these obstacles. In this review, we provide insights into the development of non-viral synthetic miRNA vectors and the promise of miRNA-based anticancer therapies, including therapeutic applications of miRNAs, challenges of vector design to overcome the delivery obstacles, and the development of miRNA delivery systems for cancer therapy. Additionally, we highlight some representative examples that give a glimpse into the current trends into the design and application of efficient synthetic systems for miRNA delivery. Overall, a better understanding of the rational design of miRNA delivery systems will promote their translation into effective clinical treatments.

Molecular-dynamics-simulation-driven design of a protease-responsive probe for in-vivo tumor imaging.

A protease-responsive probe is developed based on a molecular dynamics simulation method for the rational design of the hairpin "turn" structure of peptides. The Förster resonance energy transfer (FRET)-based probe is used for in vivo detection of legumain, a protease overexpressed in inflammation-related carcinogenesis, providing a potential method for early cancer detection and tumor imaging, and helpful information for better understanding legumain's role in tumorigenesis.