Water extract of earthworms mitigates mouse liver fibrosis by potentiating hepatic LKB1/Nrf2 axis to inhibit HSC activation and hepatocyte death.

ETHNOPHARMACOLOGICAL RELEVANCE: When left untreated, liver fibrosis (LF) causes various chronic liver diseases. Earthworms (Pheretima aspergillum) are widely used in traditional medicine because of their capacity to relieve hepatic diseases. AIM OF THE STUDY: This study aimed to explore the anti-LF effects of water extract of earthworms (WEE) and the underlying molecular mechanisms. MATERIALS AND METHODS: A CCl4-induced mouse model of LF was used to study the impact of WEE on LF in vivo. The anti-LF activity of WEE in mice was compared with that of silybin, which can be clinically applied in LF intervention and was used as a positive control. Activation of LX-2 hepatic stellate cells (HSCs) and apoptosis and ferroptosis of AML-12 hepatocytes induced by TGFß1 were used as in vitro models. RESULTS: WEE drastically improved LF in mice. WEE reduced markers of activated HSCs in mice and inhibited TGFß1-induced activation of LX-2 HSCs in vitro. Additionally, WEE suppressed CCl4-induced apoptosis and ferroptosis in mouse hepatocytes. Mechanistically, WEE induced Nrf2 to enter the nuclei of the mouse liver cells, and the hepatic levels of Nrf2-downstream antioxidative factors increased. LKB1/AMPK/GSK3ß is an upstream regulatory cascade of Nrf2. In the LF mouse model, WEE increased hepatic phosphorylated LKB1, AMPK, and GSK3ß levels. Similar results were obtained for the LX-2 cells. In AML-12 hepatocytes and LX-2 HSCs, WEE elevated intracellular Nrf2 levels, promoted its nuclear translocation, and inhibited TGFß1-induced ROS accumulation. Knocking down LKB1 abolished the impact of WEE on the AMPK/GSK3ß/Nrf2 cascade and eliminated its protective effects against TGFß1. CONCLUSIONS: Our findings reveal that WEE improves mouse LF triggered by CCl4 and supports its application as a promising hepatoprotective agent against LF. The potentiation of the hepatic antioxidative AMPK/GSK3ß/Nrf2 cascade by activating LKB1 and the subsequent suppression of HSC activation and hepatocyte apoptosis and ferroptosis are implicated in WEE-mediated alleviation of LF.

Ethanol Extract of Rosa rugosa Ameliorates Acetaminophen-Induced Liver Injury via Upregulating Sirt1 and Subsequent Potentiation of LKB1/AMPK/Nrf2 Cascade in Hepatocytes.

Acetaminophen (APAP)-induced liver injury is a common hepatic disease resulting from drug abuse. Few targeted treatments are available clinically nowadays. The flower bud of Rosa rugosa has a wide range of biological activities. However, it is unclear whether it alleviates liver injury caused by APAP. Here, we prepared an ethanol extract of Rosa rugosa (ERS) and analyzed its chemical profile. Furthermore, we revealed that ERS significantly ameliorated APAP-induced apoptosis and ferroptosis in AML-12 hepatocytes and dampened APAP-mediated cytotoxicity. In AML-12 cells, ERS elevated Sirt1 expression, boosted the LKB1/AMPK/Nrf2 axis, and thereby crippled APAP-induced intracellular oxidative stress. Both EX527 and NAM, which are chemically unrelated inhibitors of Sirt1, blocked ERS-induced activation of LKB1/AMPK/Nrf2 signaling. The protection of ERS against APAP-triggered toxicity in AML-12 cells was subsequently abolished. As expression of LKB1 was knocked down, ERS still upregulated Sirt1 but failed to activate AMPK/Nrf2 cascade or suppress cytotoxicity provoked by APAP. Results of in vivo experiments showed that ERS attenuated APAP-caused hepatocyte apoptosis and ferroptosis and improved liver injury and inflammation. Consistently, ERS boosted Sirt1 expression, increased phosphorylations of LKB1 and AMPK, and promoted Nrf2 nuclear translocation in the livers of APAP-intoxicated mice. Hepatic transcriptions of HO-1 and GCLC, which are downstream antioxidant genes of Nrf2, were also significantly increased in response to ERS. Our results collectively indicated that ERS effectively attenuates APAP-induced liver injury by activating LKB1/AMPK/Nrf2 cascade. Upregulated expression of Sirt1 plays a crucial role in ERS-mediated activation of LKB1.

Ergothioneine suppresses hepatic stellate cell activation via promoting Foxa3-dependent potentiation of the Hint1/Smad7 cascade and improves CCl4-induced liver fibrosis in mice.

Ergothioneine (EGT) is a bioactive compound derived from certain edible mushrooms. The activation of hepatic stellate cells (HSCs) is critically involved in the etiology of liver fibrosis (LF). Here, we report that in LX-2 HSCs, EGT upregulates the expression of Hint1 and Smad7 and suppresses their activation provoked by TGFß1. The EGT-triggered inhibition of HSC activation is abolished by knocking down the expression of Hint1. Overexpression of Hint1 increases Smad7 and represses TGFß1-provoked activation of LX-2 HSCs. In silico predictions unveiled that in the promoter region of the human Hint1 gene, there are two conserved cis-acting elements that have the potential to interact with the transcription factor Foxa3 termed hFBS1 and hFBS2, respectively. The knockdown of Foxa3 obviously declined Hint1 expression at both mRNA and protein levels. Transfection of Foxa3 or EGT treatment increased the activity of the luciferase reporter driven by the Hint1 promoter in an hFBS2-dependent manner. The knockdown of Foxa3 eliminated EGT-mediated upregulation of Hint1 promoter activity. Additionally, EGT triggered the nuclear translocation of Foxa3 without obviously affecting its expression level. Molecular docking analysis showed that EGT has the potential to directly interact with the Foxa3 protein. Moreover, Foxa3 played a critical role in EGT-mediated hepatoprotection. EGT modulated the Foxa3/Hint1/Smad7 signaling in mouse primary HSCs and inhibited their activation. The gavage of EGT considerably relieved CCl4-induced LF in mice. Our data provide new insights into the anti-LF activity of EGT. Mechanistically, EGT triggers the nuclear translocation of Foxa3 in HSCs, which promotes Hint1 transcription and subsequently elevates Smad7.

Theaflavine inhibits hepatic stellate cell activation by modulating the PKA/LKB1/AMPK/GSK3ß cascade and subsequently enhancing Nrf2 signaling.

Activation of hepatic stellate cells (HSCs) constitutes a crucial etiological factor leading to liver fibrosis. Theaflavine (TF) is a characteristic bioactive compound in fermented tea. Here, we found that TF attenuated the activation of LX-2 HSCs induced by transforming growth factor-ß1 (TGF-ß1). TF potentiated nuclear factor erythroid 2-related Factor 2 (Nrf2) signaling. Knockdown of Nrf2 abrogated TF-mediated resistance to TGF-ß1. Liver kinase B1 (LKB1), AMP-activated kinase (AMPK), and glycogen synthase kinase-3ß (GSK3ß) are upstream regulators of Nrf2. TF modulated the LKB1/AMPK/GSK3ß axis. Inhibition of AMPK or knockdown of LKB1 crippled TF-mediated potentiation of Nrf2. Protein kinase A (PKA) catalyzes LKB1 phosphorylation. In LX-2 cells, TF increased the LKB1/PKA interaction without affecting their contents. Inhibition of PKA abolished TF-mediated potentiation of LKB1/Nrf2 and abrogated the inhibitory effects of TF on their activation. TF also enhanced direct binding between purified catalytic subunit α of PKA (PKA-Cα) and LKB1 proteins in vitro. Molecular docking indicated that TF showed binding activity with both LKB1 and PKA-Cα proteins. In mouse primary HSCs, TF elevated LKB1/PKA-Cα binding, boosted LKB1 phosphorylation, potentiated Nrf2 and suppressed their spontaneous activation. PKA inhibition or LKB1 knockdown eliminated TF-mediated induction of Nrf2 and suppression of HSC activation. Furthermore, TF considerably alleviated CCl4-induced mouse liver fibrosis. In mouse livers, TF increased the LKB1/PKA-Cα interaction, upregulated LKB1 phosphorylation and modulated its downstream AMPK/GSK3ß/Nrf2 cascade. Our findings collectively indicated that TF suppresses HSC activation. Mechanistically, TF elevated the LKB1/PKA interaction in HSCs, which increased LKB1 phosphorylation and subsequently modulated the downstream AMPK/GSK3ß/Nrf2 axis.

Rapid detection of TNP based on a commercial fluorescent probe.

A simple, rapid and low cost sensing method for the fluorescent detection of TNP in 100% aqueous media was successfully developed based on a commercial probe. Under the non-covalent interactions between TNP with probe, a ratiometric output signal was achieved, which can be applied in real samples test.

An amino acidic adjuvant to augment cryoinjury of MCF-7 breast cancer cells.

One of the major challenges in cryosurgery is to minimize incomplete cryodestruction near the edge of the iceball. In the present study, the feasibility and effectiveness of an amino acidic adjuvant, glycine was investigated to enhance the cryodestruction of MCF-7 human breast cancer cell at mild freezing/thawing conditions via eutectic solidification. The effects of glycine addition on the phase change characteristics of NaCl-water binary mixture were investigated with a differential scanning calorimeter and cryo-macro/microscope. The results confirmed that a NaCl-glycine-water mixture has two distinct eutectic phase change events - binary eutectic solidification of water-glycine, and ternary eutectic solidification of NaCl-glycine-water. In addition, its effects on the cryoinjury of MCF-7 cells were investigated by assessing the post-thaw cellular viability after a single freezing/thawing cycle with various eutectic solidification conditions due to different glycine concentrations, end temperatures and hold times. The viability of MCF-7 cells in isotonic saline supplemented with 10% or 20% glycine without freezing/thawing remained higher than 90% (n=9), indicating no apparent toxicity was induced by the addition of glycine. With 10% glycine supplement, the viability of the cells frozen to -8.5 degrees C decreased from 85.9+/-1.8% to 38.5+/-1.0% on the occurrence of binary eutectic solidification of glycine-water (n=3 for each group). With 20% glycine supplement, the viability of the cells frozen to -8.5 degrees C showed similar trends to those with 10% supplement. However, as the end temperature was lowered to -15 degrees C, the viability drastically decreased from 62.5+/-2.0% to 3.6+/-0.7% (n=3 for each group). The influences of eutectic kinetics such as nucleation temperature, hold time and method were less significant. These results imply that the binary eutectic solidification of water-glycine can augment the cryoinjury of MCF-7 cells, and the extent of the eutectic solidification is significant.

De novo formation of centrosomes in vertebrate cells arrested during S phase.

The centrosome usually replicates in a semiconservative fashion, i.e., new centrioles form in association with preexisting "maternal" centrioles. De novo formation of centrioles has been reported for a few highly specialized cell types but it has not been seen in vertebrate somatic cells. We find that when centrosomes are completely destroyed by laser microsurgery in CHO cells arrested in S phase by hydroxyurea, new centrosomes form by de novo assembly. Formation of new centrosomes occurs in two steps: approximately 5-8 h after ablation, clouds of pericentriolar material (PCM) containing gamma-tubulin and pericentrin appear in the cell. By 24 h, centrioles have formed inside of already well-developed PCM clouds. This de novo pathway leads to the formation of a random number of centrioles (2-14 per cell). Although clouds of PCM consistently form even when microtubules are completely disassembled by nocodazole, the centrioles are not assembled under these conditions.