AdipoR1/AdipoR2 dual agonist recovers nonalcoholic steatohepatitis and related fibrosis via endoplasmic reticulum-mitochondria axis.

Chronic nonalcoholic steatohepatitis (NASH) is a metabolic disorder that often leads to liver fibrosis, a condition with limited therapy options. Adiponectin is an adipocytokine that regulates glucose and lipid metabolism via binding to its receptors AdipoR1 and AdipoR2, and AdipoRs signaling is reported to enhance fatty acid oxidation and glucose uptake. Here, we synthesize and report an adiponectin-based agonist JT003, which potently improves insulin resistance in high fat diet induced NASH mice and suppresses hepatic stellate cells (HSCs) activation in CCl4 induced liver fibrosis. Mechanistic studies indicate that JT003 simultaneously stimulates AdipoR1- and AdipoR2- mediated signaling pathways as well as the PI3K-Akt pathway. Moreover, JT003 treatment significantly improves ER-mitochondrial axis function, which contributes to the reduced HSCs activation. Thus, the AdipoR1/AdipoR2 dual agonist improves both NASH and fibrosis in mice models, which provides the pharmacological and biological foundation for developing AdipoRs-based therapeutic agents on liver fibrosis.

RAP-8 ameliorates liver fibrosis by modulating cell cycle and oxidative stress.

AIMS: The rapeseed protein derived peptide DHNNPQIR (named as RAP-8) has been previously reported to possess antioxidant activity and alleviate liver fibrosis. The purpose of the present study was to investigate the potential crucial pathways involved in ameliorating liver fibrosis of RAP-8. MAIN METHODS: Next-generation sequencing of messenger RNA (RNA-Seq) analysis of the fibrotic and RAP-8 treated mice was performed. Western blot, qPCR and flow cytometry detection analysis were conducted to measure cell cycle and oxidative stress in LX-2 cells and liver samples. KEY FINDINGS: 588 overlapped differentially expressed genes were obtained from a batch of genes RAP-8 altered. Gene Ontology enrichment analysis revealed that changes in the most significant modules were mainly enriched in cell division, nuclear division and mitotic cell cycle process, while alterations in Kyoto Encyclopedia of Genes and Genomes were mainly enriched in cell cycle. Thereafter, according to the co-expression network analysis, the regulations of three core genes (Cenpp, Cyp2c55, Serpinh1) were verified that might be targets for treating liver fibrosis. Furthermore, through experimental verification, we demonstrated that RAP-8 induced cell cycle arrest and prevents oxidation stress. SIGNIFICANCE: As a promising therapeutic candidate for hepatic fibrosis treating, RAP-8 exhibited anti-fibrotic effects via exerting cell cycle arrest and inhibiting oxidative stress.

Site-specific labeling of an anti-MUC1 antibody: probing the effects of conjugation and linker chemistry on the internalization process.

Antibody-drug conjugates (ADCs) have recently received enormous attention as an attractive approach for cancer therapy. Although ADC design has been believed to be important for the relative efficacy of ADCs, it remains unexplored how the structural characteristics of ADCs would impact the internalization process and intracellular trafficking of the molecules. Herein, we report our efforts in investigating the cellular endocytosis implications of the conjugation and linker chemistry in designing antibody-based agents. A series of anti-MUC1 single-chain variable fragment (scFv-SM3) conjugates were designed with unique structural characteristics ranging from conjugation methods, sites of attachment and linker chemistry. In vitro confocal imaging showed that both random lysine-conjugation and site-specific conjugation, including C-terminus modification or internal site conjugation, could afford antibody conjugates with similar binding affinity and cellular uptake to target-expressing cells. Time-course internalization studies demonstrated that SM3-conjugates with short polyethylene glycol linkers outcompeted those that lack any hydrophilic linkers for higher cellular uptake and faster internalization rate. The SM3-conjugates with the highest affinity and internalization rate were also tested in mouse xenograft models using MUC1-overexpressing tumor cells. Our results indicate that the linker and conjugation chemistry play an important role in the internalization process of antibody conjugates, and this in turn could impact the therapeutic effects of ADCs.

Rapeseed Protein-Derived Antioxidant Peptide RAP Ameliorates Nonalcoholic Steatohepatitis and Related Metabolic Disorders in Mice.

Rapeseed protein hydrolysates have recently shown in vitro antioxidant and anti-inflammatory activities. However, scant data exist about their in vivo activities. Here, we report that the peptide DHNNPQIR (hereinafter referred to as RAP-8), a bioactive peptide originated from rapeseed protein, exhibits excellent in vivo efficacy in mouse models of nonalcoholic steatohepatitis (NASH) and hepatic fibrosis. We demonstrated that RAP-8 significantly reduced hepatic steatosis and improved insulin resistance and lipid metabolism. Furthermore, RAP-8 showed markedly reduced hepatic inflammation, fibrosis, liver injury, and metabolic deterioration. In particular, RAP-8 directly suppressed fibrosis-associated gene expression, including α-smooth muscle actin (α-Sma) and collagen type I (Col-1α) in the liver of mice in vivo. In addtion, RAP-8 significantly decreased macrophage infiltration and reduced pro-inflammatory cytokines secretion. Finally, we found that RAP-8 administration significantly decreased oxidative stress-induced apoptosis in liver injury induced by CCl4. Therefore, our results suggest that RAP-8 could be available for treatment of NASH and NASH-related metabolic disorders as a potential therapeutic candidate.

Phosphine-Catalyzed Enantioselective [4 + 2] Cycloaddition-Semipinacol-Type-Rearrangement Reaction of Morita-Baylis-Hillman Carbonates.

The chiral phosphine-triggered electrophilic ylide intermediate for a Morita-Baylis-Hillman carbonates activation strategy provides a promising method for the design of organocatalytic intermolecular higher-order annulation processes.

Direct ß-selectivity of α,ß-unsaturated γ-butyrolactam for asymmetric conjugate additions in an organocatalytic manner.

The ß-selective asymmetric addition of γ-butyrolactam with cyclic imino esters catalyzed by a bifunctional chiral tertiary amine has been developed, which provides an efficient access to optically active ß-position functionalized pyrrolidin-2-one derivatives in both high yield and enantioselectivity (up to 78% yield and 95 : 5 er). This is the first catalytic method to access chiral ß-functionalized pyrrolidin-2-one via a direct organocatalytic approach.