rAj-HRP30 is a recombinant peptide derived from the wild-type rAj-HRP of Apostichopus japonicus through a gene-shortening mutation. It has a high histidine content (53.3% in its primary structure) and a molecular weight of 3.919 kDa, classifying it as a histidine-rich peptide. The literature reports indicate that human histidine-rich peptides exhibit antitumor activity. Previous research by our group demonstrated similar properties in rAj-HRP, the precursor of rAj-HRP30. Therefore, this study used Panc01 (human) and Panc02 (mouse) cells—highly malignant models with limited targeted therapies—to investigate the antitumor activity and mechanisms of rAj-HRP30 and evaluate its potential for pancreatic cancer treatment. This study designed a gene-shortening strategy for rAj-HRP and artificially synthesized the gene sequence of rAj-HRP30. The cDNA sequence of rAj-HRP30 was cloned into the pET23b vector, and the recombinant plasmid pET23b-HRP30 was transformed into E. coli BL21 for expression. Following IPTG induction, the recombinant peptide was purified using nickel ion affinity chromatography, yielding rAj-HRP30 with a purity exceeding 95%. rAj-HRP30 markedly inhibited the adhesion, migration, and invasion of Panc01 and Panc02 cells. It also disrupted cellular morphology and cytoskeletal structure while inducing apoptosis. These effects were dose-dependent. After confirming the in vitro anticancer activity of rAj-HRP30, this study employed Panc02 cells as a model to investigate its inhibitory mechanisms using Western blot analysis. The results revealed that rAj-HRP30 reduced FGFR1 expression in Panc02 cells and inhibited the downstream FYN and FAK signaling pathways, subsequently blocking the PI3K/AKT signaling and apoptosis pathways. In the apoptotic pathway, rAj-HRP30 was able to downregulate the expression of Bcl-2, Caspase-9, Caspase-3, Caspase-7, and PARP1 and upregulate the expression of Bax, cleaved Caspase-9, cleaved Caspase-3, cleaved Caspase-7, and cleaved-PARP1 to induce apoptosis in Panc02 cells. Furthermore, rAj-HRP30 also downregulated the expression of MMP2 and MMP9, thereby inhibiting the migration and invasion of Panc02 cells. Conclusion: rAj-HRP30 exhibits significant inhibitory effects on pancreatic ductal adenocarcinoma Panc01 and Panc02 cells in vitro. Its mechanism involves FGFR1-related signaling and apoptosis pathways. rAj-HRP30 shows promise as a therapeutic agent targeting FGFR for pancreatic cancer.

BACKGROUND:Acetaminophen-induced hepatotoxicity remains a clinical challenge with limited targeted therapeutic options. While recent advances have identified blood‒biliary barrier disruption as a critical pathogenic mechanism, effective interventions preserving this barrier are notably lacking. Albiflorin, a key bioactive constituent of Paeonia lactiflora Pall., exhibits unique bile acid modulation along with antioxidant and anti-inflammatory activities, suggesting its potential efficacy in preventing acetaminophen-induced liver injury. However, its specific role and underlying mechanisms in alleviating acetaminophen-induced hepatotoxicity remain unclear. OBJECTIVE:This study's objective was to examine the pharmacological effects and primary molecular mechanisms of albiflorin in alleviating acetaminophen-induced liver injury. METHODS:An acetaminophen-induced liver injury mouse model was created using a 300 mg/kg dose of acetaminophen. The hepatoprotective effects of albiflorin were assessed through histological and biochemical analyses. Blood‒biliary barrier integrity was evaluated via Evans blue dye tests, immunofluorescence, and bile acid assays. Transcriptomic analysis, gene overexpression and interference techniques, and ChIP‒qPCR were employed to explore the molecular mechanisms underlying the protective effects of albiflorin. RESULTS:Albiflorin significantly reduced acetaminophen-induced liver injury, as evidenced by improved biochemical profiles and hepatocyte morphology. It also prevented increases in blood‒biliary barrier permeability and bile acid levels. RNA sequencing identified 3,184 differentially expressed genes, revealing critical pathways involved in maintaining blood‒biliary barrier integrity. AF reversed the acetaminophen-induced changes in the expression of genes related to the blood‒biliary barrier, particularly occludin, claudin 5, ABCG5, and ABCG8. Albiflorin protected the blood‒biliary barrier and mitigated acetaminophen-induced liver injury by enhancing ATF3 protein stability, with ATF3 identified as a critical mediator of these protective effects. CONCLUSION:This study provides pioneering evidence that albiflorin protects against acetaminophen-induced liver injury by interacting with ATF3 to regulate blood-biliary barrier proteins and maintain the integrity of the blood-biliary barrier. These findings deepen our understanding of the role of the blood‒biliary barrier in liver diseases and suggest a therapeutic strategy for drug overdose-induced liver injury.

Colon cancer is a common and lethal malignancy, ranking second in global cancer-related mortality, highlighting the urgent need for novel targeted therapies. The sea cucumber (Apostichopus japonicus) is a marine organism known for its medicinal properties. After conducting a bioinformatics analysis of the cDNA library of Apostichopus japonicus, we found and cloned a cDNA sequence encoding histidine-rich peptides, and the recombinant peptide was named rAj-HRP. Human histidine-rich peptides are known for their anti-cancer properties, raising questions as to whether rAj-HRP might exhibit similar effects. To investigate whether rAj-HRP can inhibit colon cancer, we used human colon cancer HCT116 cells as a model and studied the tumor suppressive activity in vitro and in vivo. The results showed that rAj-HRP inhibited HCT116 cell proliferation, migration, and adhesion to extracellular matrix (ECM) proteins in vitro. It also disrupted the cytoskeleton and induced apoptosis in these cells. In vivo, rAj-HRP significantly inhibited the growth of HCT116 tumors in BALB/c mice, reducing tumor volume and weight without affecting the body weight of the tumor-bearing mice. Western blot analysis showed that rAj-HRP inhibited HCT116 cell proliferation and induced apoptosis by upregulating BAX and promoting PARP zymogen degradation. Additionally, rAj-HRP inhibited HCT116 cell adhesion and migration by reducing MMP2 levels. Further research showed that rAj-HRP downregulated EGFR expression in HCT116 cells and inhibited key downstream molecules, including AKT, P-AKT, PLCγ, P38 MAPK, and c-Jun. In conclusion, rAj-HRP exhibits significant inhibitory effects on HCT116 cells in both in vitro and in vivo, primarily through the EGFR and apoptosis pathways. These findings suggest that rAj-HRP has the potential as a novel targeted therapy for colon cancer.

The emergence of extracellular vesicles (EVs), which are natural lipid bilayer membrane structures facilitating intercellular substance and information exchange, has sparked innovative approaches in drug development and carrier enhancement. Plant-derived EVs notably offer advantages including low preparation cost, low immunogenicity, flexible drug delivery, high stability, good tissue permeability, and high inherent medicinal value compared to their animal-derived counterparts. Despite these promising attributes, the research on plant-derived EVs remains fragmented and lacks comprehensive synthesis. This review aims to address this gap by summarizing the isolation methods, biological characteristics, and storage techniques of plant-derived EVs. Additionally, we explore the potential of plant-derived EVs as therapeutic agents and drug carriers for treating various diseases. Finally, we delineate the current impediments to plant-derived EV development and highlight future research directions. By providing a detailed overview, we hope to facilitate further research and application in this emerging field.

Background: Cerebral ischemia-reperfusion injury (CI/RI) is a complex process leading to neuronal damage and death, with mitophagy implicated in its pathogenesis. However, the significance of mitophagy in CI/RI remains debated. Hypothesis: We hypothesized that TRIM25 reduces ATAD3A expression by ubiquitinating ATAD3A, promoting mitophagy via the PINK1/Parkin pathway, and aggravating CI/RI. Study design: Rat middle cerebral artery occlusion (MCAO) followed by reperfusion and oxygen-glucose deprivation and reoxygenation (OGD/R) in PC12 cells were used as animal and cell models, respectively. Methods: To evaluate the success of the CI/R modeling, TTC and HE staining were employed. The determination of serum biochemical indexes was carried out using relative assay kits. The Western Blot analysis was employed to assess the expression of ATAD3A, TRIM25, as well as mitophagy-related proteins (PINK1, Parkin, P62, and LC3II/LC3I). The mRNA levels were detected using QRT-PCR. Mitochondrial membrane potential was assessed through JC-1 staining. Mitosox Red Assay Kit was utilized to measure mitochondrial reactive oxygen species levels in PC12 cells. Additionally, characterization of the mitophagy structure was performed using transmission electron microscopy (TEM). Results: Our findings showed down-regulation of ATAD3A and up-regulation of TRIM25 in both in vivo and in vitro CI/RI models. Various experimental techniques such as Western Blot, JC-1 staining, Mitosox assay, Immunofluorescence assay, and TEM observation supported the occurrence of PINK1/Parkin signaling pathwaymediated mitophagy in both models. ATAD3A suppressed mitophagy, while TRIM25 promoted it during CI/RI injury. Additionally, the results indicated that TRIM25 interacted with and ubiquitinated ATAD3A via the proteasome pathway, affecting ATAD3A protein stability and expression. Conclusion: TRIM25 promoted Pink1/Parkin-dependent excessive mitophagy by destabilizing ATAD3A, exacerbating CI/RI. Targeting TRIM25 and ATAD3A may offer therapeutic strategies for mitigating CI/RI and associated neurological damage.

rAj-Tspin, a soluble recombinant peptide from Apostichopus japonicus, can inhibit the integrin β1 (ITGB1)/FAK/AKT signaling pathway in hepatocellular carcinoma (HCC) via cell epithelial–mesenchymal transition (EMT) and apoptosis. Zyxin (ZYX) is a focal adhesion protein that is considered a novel mediator of EMT and apoptosis. However, the inhibitory mechanisms of rAj-Tspin in HCC and whether it is related to ZYX are unclear. We examined the antitumor effect of rAj-Tspin on the Huh7 human HCC cell line and on a nude mouse model generated via subcutaneous injection or orthotopic intrahepatic transplantation of Huh7 cells. Our results revealed that rAj-Tspin strikingly reduced the viability and promoted the apoptosis of Huh7 cells and inhibited HCC tumor growth in nude mice. rAj-Tspin inhibited ITGB1 and ZYX protein expression in vivo and in vitro in a dose-dependent manner. Mechanistically, the FAK/AKT signaling pathway and the proliferation and invasion of HCC cells were suppressed upon ITGB1 and ZYX knockdown. Moreover, the effect of ITGB1 overexpression on the growth of HCC cells was inhibited by rAj-Tspin. In contrast, the promoting effect of ITGB1 overexpression could be inhibited by ZYX knockdown. ZYX knockdown had no effect on ITGB1 expression. These findings suggest that ZYX is required for the indispensable role of ITGB1 in rAj-Tspin‐alleviated HCC and provide an important therapeutic target for HCC. In summary, the anti-HCC effect of rAj-Tspin potentially involves the regulation of the ITGB1/ZYX/FAK/AKT pathway, which in turn impacts EMT and apoptosis.

rLj-RGD3, a new member of the RGD (Arginine-Glycine-Aspartate)-motif toxin protein family obtained from Lampetra japonica by means of recombinant DNA techniques, has been demonstrated to be a platelet fibrinogen receptor antagonist and holds potential as a drug candidate for a specific indication. The present article reports an innovative validated highly sensitive and specific biotin-avidin enzyme linked immunosorbent assay (BA-ELISA) to provide a bio-analytical method for pharmacokinetic (PK) studies of rLj-RGD3. The concentration of picogram level rLj-RGD3 in rat plasma was measured using the developed double sandwich BA-ELISA assay, which used two mouse anti-rLj-RGD3 monoclonal antibodies that recognize different epitopes for capture and detection. This method was verified to be highly specific (blank plasma did not interfere with detection), precise (RSD <15%), and accurate (86%-113%). Absolute recovery was in the 94%-119% range. The calibration curve showed good linearity within the 50 to 1600 pg/mL range. The LOQ was as low as 50 pg/mL. The above validated assay was successfully employed to assess PK of rLj-RGD3 in rats. After i.v. and s.c. dosing with 30 μg/kg, the rLj-RGD3 plasma concentration declined bi-exponentially with time. This decay was best fitted to a two-compartment model. In conclusion, the BA-ELISA method described here meets all requirements for PK studies of rLj-RGD3 with an effective pharmacological dose in the μg/kg BW range.

HAC is a rare extrahepatic malignancy histologically resembling hepatocellular carcinoma which can occur in many organs.Especially for cases carrying HAC within the stomach or duodenum, we found that typically HAC only happened in either of them and there was no case exhibiting HAC in both sites.Here, we presented a case having HAC in both sites and to our knowledge, this is the first report presenting HAC in these two distinct organs simultaneously.Firstly, a 57-year-old male was tested for increased serum alpha-fetoprotein level.Following PET-CT images showed strengthened FDG uptake in the gastric antrum and proximal duodenum.Esophagogastroduodenoscopy also revealed an irregular lump at the gastric antrum and a deep ulceration at the duodenum bulb.Next, an immunohistochemistry panel confirmed the diagnosis of HAC.Finally, genetic tests were performed on this patient after the clinical diagnosis of HAC to reveal the molecular etiology.

Alcohol-associated liver disease (ALD) is a liver system disease caused by alcohol abuse, and it involves complex processes ranging from steatosis to fibrosis, cirrhosis and hepatocellular carcinoma. Steatosis and inflammation are the main phenomena involved in ALD. Ubiquitin-specific protease 22 (USP22) plays an important role in liver steatosis; however, its functional contribution to ALD remains unclear. USP22-silenced mice were fed a Lieber-DeCarli liquid diet. AML-12 and HEK293T cells were used to detect the interaction between USP22 and BRD4. Here, we report that hepatic USP22 expression was dramatically upregulated in mice with ALD. Inflammation and steatosis were significantly ameliorated following USP22 silencing in vivo, as indicated by decreased IL-6 and IL-1β levels. We further showed that the overexpression of USP22 increased inflammation, while knocking down BRD4 suppressed the inflammatory response in AML-12 cells. Notably, USP22 functioned as a BRD4 deubiquitinase to facilitate BRD4 inflammatory functions. More importantly, the expression levels of USP22 and BRD4 in patients with ALD were significantly increased. In conclusion, USP22 acts a key pathogenic factor in ALD by deubiquitinating BRD4, which facilitates the inflammatory response and aggravates ALD.

The treatment options for cancer of unknown primary (CUP) are challenging due to the lack of knowledge about the primary sites, often resulting in a poor prognosis. The emerging next-generation sequencing (NGS) technique has provided a reliable approach to facilitate tumor primary site prediction and targetable gene alteration identification for CUP patients. In this report, we described a 63-year-old female patient who experienced recurrent CUP. NGS-based genetic profiling results revealed a pathogenic germline BRCA1 R71K mutation. Accordingly, the patient received the poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitor olaparib treatment and demonstrated a favorable response to this treatment. Our case suggests that NGS holds great promise for providing improved diagnosis and treatment options to patients with CUP, warranting further clinical investigation.

系统性红斑狼疮(SLE)患者常伴有消化系统受累表现，但以消化系统症状首发的病例相对少见，因症状无特异性，易被误诊、误治。该例SLE患者以急性腹膜炎为首发表现，经多学科协作会诊，最终确诊。现分享诊治思路，为临床类似病例提供参考和借鉴。

目的:探讨仅表现为恶性胸腹腔积液的间变性淋巴瘤激酶（ALK）阴性的间变性大细胞淋巴瘤（ALCL）的临床病理特征。方法:回顾性分析大连医科大学附属第二医院1例仅表现为恶性胸腹腔积液的ALK - ALCL患者的临床资料，并复习相关文献。 结果:该患者临床表现仅为大量胸腹腔积液，全身无浅表淋巴结肿大，影像学检查亦未发现实质性肿物，在对腹腔积液包埋沉渣进行免疫组织化学检查后，结合细胞学涂片，腹腔积液液基制片以及流式细胞术分析和TCR基因重排检测结果，诊断为ALK-ALCL。给予化疗后，腹腔积液立即消失，Ⅰ期化疗后恢复良好，2016年完成8个周期化疗后停药。随访至截稿前，患者停药后首次因腹腔积液入院，充分引流后，症状即缓解，亦未发现实体肿物，维持西达苯胺治疗，随诊观察。结论:腹腔积液诊断为ALCL少见，无实体肿瘤者更为罕见，需和原发渗出性淋巴瘤、弥漫性大B细胞淋巴瘤、间皮瘤等进行鉴别；部分ALK - ALCL预后良好。

We previously found that inhibition of p66Shc confers protection against hepatic stellate cell (HSC) activation during liver fibrosis. However, the effect of p66Shc on HSC proliferation, as well as the mechanism by which p66Shc is modulated, remains unknown. Here, we elucidated the effect of p66Shc on HSC proliferation and evaluated microRNA (miRNA)-p66Shc-mediated reactive oxidative species (ROS) generation in liver fibrosis. An in vivo model of carbon tetrachloride (CCl4)-induced liver fibrosis in rats and an LX-2 cell model were developed. p66Shc expression was significantly upregulated in rats with CCl4-induced liver fibrosis and in human fibrotic livers. Additionally, p66Shc knockdown in vitro attenuated mitochondrial ROS generation and HSC proliferation. Interestingly, p66Shc promoted HSC proliferation via β-catenin dephosphorylation in vitro. MicroRNA (miR)-203a-3p, which was identified by microarray and bioinformatics analyses, directly inhibited p66Shc translation and attenuated HSC proliferation in vitro. Importantly, p66Shc was found to play an indispensable role in the protective effect of miR-203a-3p. Furthermore, carnosic acid (CA), the major antioxidant compound extracted from rosemary leaves, protected against CCl4-induced liver fibrosis through the miR-203a-3p/p66Shc axis. Collectively, these results suggest that p66Shc, which is directly suppressed by miR-203a-3p, is a key regulator of liver fibrosis. This finding may lead to the development of therapeutic targets for liver fibrosis.

The toxic RGD peptide, rLj-RGD4, characterized by 4 (Arg-Gly-Asp) (RGD) motifs, is a novel mutant of rLj-RGD3 from the salivary gland of Lampetra japonica. Our previous study showd that rLj-RGD3 exerts a protective effect against cerebral ischemia injury in rats. Through the induction of middle cerebral artery occlusion/reperfusion (MCAO) injuries in rats, the present study investigated the effects and the mechanism through which rLj-RGD4 protects against ischemic stroke. In rats, treatment with rLj-RGD4 2 h after MCAO enhanced survival rate, improved movement and ameliorated the severity of brain infarction and apoptosis by diminishing pathological changes. This study demonstrated that rLj-RGD4 can reverse the downregulation of Bcl2, and the upregulation of Caspase-3. Mechanistic studies showed that rLj-RGD4 upregulated the expression levels of FAK, p-FAK, PI3K and p-Akt. In contrast, caspase-3 expression was inhibited. These results showed that rLj-RGD4 may reduce cerebral ischemia-reperfusion injury.

Purpose Our study aimed to investigate the antitumor effects of rAj-Tspin on BEL-7402 hepatocellular carcinoma cells and to explore the underlying mechanism. Method For the in vivo experiment, BEL-7402 cells were inoculated subcutaneously into the axilla of nude mice to generate a BEL-7402 cell-bearing model, and model mice were then treated with different doses of rAj-Tspin. A CCK-8 assay was used to evaluate the in vitro viability of BEL-7402 and LO2 cells after treatment with different concentrations of rAj-Tspin. The effects of rAj-Tspin on BEL-7402 cell apoptosis, migration and invasion were evaluated by AO/EB and Hoechst fluorescent staining and by scratch and Transwell assays, and the tumor-suppressive mechanism of rAj-Tspin was explored by Western blotting. Results rAj-Tspin suppressed the proliferation of BEL-7402 cells with an IC50 of 0.89 μM. The results of both microscopic analysis and Western blotting suggested that rAj-Tspin induced the apoptosis of BEL-7402 cells through a mitochondria-dependent pathway. Furthermore, rAj-Tspin disrupted EMT; this disruption ultimately caused BEL-7402 cells to lose their shape and decreased their migration and invasion. Moreover, rAj-Tspin might inhibit the proliferation and metastasis of BEL-7402 cells through the ITGB1-FAK-AKT pathway. Conclusion rAj-Tspin exerts an antitumor effect through the ITGB1-FAK-Akt signaling pathway and exhibits low toxicity at an effective dose.

Saccharides from Arctium lappa. L. root (ALR-S) is a high-purity fructosaccharide separated from the medicinal plant Arctium lappa. L. root. These compounds showed many pharmacological effects in previous studies. In the present study, the antithrombotic effects of ALR-S in arterial thrombosis via inhibiting platelet adhesion and rebalancing thrombotic and antithrombotic factor expression and secretion were found in rats and human aortic endothelial cells (HAECs). This study also showed that inhibition of oxidative stress (OS), which is closely involved in the expression of coagulation- and thrombosis-related proteins, was involved in the antithrombotic effects of ALR-S. Furthermore, studies using FeCl3-treated HAECs showed that ALR-S induced the abovementioned effects at least partly by blocking the ERK/NF-κB pathway. Moreover, U0126, a specific inhibitor of ERK, exhibited the same effects with ALR-S on a thrombotic process in FeCl3-injured HAECs, suggesting the thrombotic role of the ERK/NF-κB pathway and the antithrombotic role of blocking the ERK/NF-κB pathway by ALR-S. In conclusion, our study revealed that the ERK/NF-κB pathway is a potential therapeutic target in arterial thrombosis and that ALR-S has good characteristics for the cure of arterial thrombosis via regulating the ERK/NF-κB signaling pathway.

p66Shc, a master regulator of mitochondrial reactive oxygen species (mtROS), is a crucial mediator of hepatocyte oxidative stress. However, its functional contribution to acetaminophen (APAP)-induced liver injury and the mechanism by which it is modulated remain unknown. Here, we aimed to assess the effect of p66Shc on APAP-induced liver injury and to evaluate if circular RNA (circRNA) functions as a competitive endogenous RNA (ceRNA) to mediate p66Shc in APAP-induced liver injury. p66Shc-, miR-185-5p-, and circ-CBFB-silenced mice were injected with APAP. AML12 cells were transfected with p66Shc, miR-185-5p, and circ-CBFB silencing or overexpression plasmids or siRNAs prior to APAP stimulation. p66Shc was upregulated in liver tissues in response to APAP, and p66Shc silencing in vivo protected mice from APAP-induced mitochondrial dynamics perturbation and liver injury. p66Shc knockdown in vitro attenuated mitochondrial dynamics and APAP-induced hepatocyte injury. Mechanically, p66Shc perturbs mitochondrial dynamics partially by inhibiting OMA1 ubiquitination. miR-185-5p, which directly suppressed p66Shc translation, was identified by microarray and bioinformatics analyses, and its overexpression attenuated mitochondrial dynamics and hepatocyte injury in vitro. Furthermore, luciferase, pull-down and RNA immunoprecipitation assays demonstrated that circ-CBFB acts as a miRNA sponge of miR-185-5p to mediate p66Shc in APAP-induced liver injury. circ-CBFB knockdown also alleviated APAP-induced mitochondrial dynamics perturbation and hepatocyte injury. More importantly, we found that the protective effects of circ-CBFB knockdown on p66Shc, mitochondrial dynamics and liver injury were abolished by miR-185-5p inhibition both in vivo and in vitro. In conclusion, p66Shc is a key regulator of APAP-induced liver injury that acts by triggering mitochondrial dynamics perturbation. circ-CBFB functions as a ceRNA to regulate p66Shc during APAP-induced liver injury, which may provide a potential therapeutic target.

This study aimed to investigate the prognostic value and molecular mechanism of DDX39 and its effector SNAIL in melanoma. First, overexpression of DDX39 in melanoma, which was identified by database analysis, was further validated in patient tissues. Cell growth, cell cycle, cell migration, and cell invasion assays were then performed to evaluate the effects of downregulated DDX39 on the melanoma cell proliferation and aggressiveness. The same approaches were also used to reveal the cooperation of the transcription factor SNAIL with DDX39 to promote the aggressiveness of melanoma cells. We found that the expression of DDX39 was significantly upregulated in melanoma tissue compared to pigmented nevus tissue, and it was positively correlated with the clinical stage defined by the American Joint Committee on Cancer (AJCC) and the prognosis. Downregulation of DDX39 in melanoma cells was found to significantly inhibit cell proliferation, increase G2/M cell cycle arrest, enhance caspase-mediated cell apoptosis, and suppress cell invasion and migration. In addition, we demonstrated that the overexpression of SNAIL could restore the cell growth and aggressiveness impaired by DDX39 RNA interference. Immunohistochemical staining also showed a positive correlation between DDX39 overexpression and SNAIL overexpression in melanoma tissues, suggesting that SNAIL is one of the effectors activated by DDX39. In summary, the overexpression of DDX39 and SNAIL was positively related to the poor prognosis of melanoma patients and the increased aggressiveness of melanoma cells. Our study provides valuable evidence regarding the prognostic value of DDX39 and SNAIL as well as their potential as novel therapeutic targets for treating melanoma patients.

Background: p66Shc is a redox enzyme that mediates mitochondrial reactive oxygen species (ROS) generation. p66Shc inhibition confers protection against liver injury, however, its functional contribution to liver fibrosis remains unclear. The aim of this study is to explore the involvement of p66Shc in liver fibrosis and underlying mechanism of p66Shc by focusing on mitochondrial ROS. Methods: p66Shc-silenced mice were injected with carbon tetrachloride (CCl4). Primary hepatic stellate cells (HSCs) were performed with p66Shc silencing or overexpression prior to TGF-β1 stimulation. Results: p66Shc expression was progressively elevated in mice with CCl4-induced liver fibrosis, and p66Shc silencing in vivo significantly attenuated fibrosis development, reducing liver damage, oxidative stress and HSC activation, indicated by the decreased α-SMA, CTGF and TIMP1 levels. Furthermore, in primary HSCs, p66Shc-mediated mitochondrial ROS production played a vital role in mitochondrial morphology and cellular metabolism. Knockdown of p66Shc significantly inhibited mitochondrial ROS production and NOD-like receptor protein 3 (NLRP3) inflammasome activation, which were closely associated with HSC activation, indicated by the decreased α-SMA, CTGF and TIMP1 levels. However, p66Shc overexpression exerted the opposite effects, which were suppressed by a specific mitochondrial ROS scavenger (mito-TEMPO). More importantly, p66Shc expression was significantly increased in human with liver fibrosis, accompanied by NLRP3 inflammasome activation. Conclusions: p66Shc is a key regulator of liver fibrosis by mediating mitochondrial ROS production, which triggers NLRP3 inflammasome activation.