Hepatitis B doubly spliced protein (HBDSP) promotes hepatocellular carcinoma cell apoptosis via ETS1/GATA2/YY1-mediated p53 transcription.

IMPORTANCE: Hepatitis B virus (HBV) spliced variants are associated with viral persistence or pathogenicity. Hepatitis B doubly spliced protein (HBDSP), which has been previously reported as a pleiotropic transactivator protein, can potentially serve as an HBV virulence factor. However, the underlying mechanisms of HBDSP in HBV-associated liver diseases remain to be elucidated. In this study, we revealed that HBDSP promotes cellular apoptosis and induces wt-p53-dependent apoptotic signaling pathway in wt-p53 hepatocellular cells by transactivating p53 transcription, and increases the release of HBV progeny. Therefore, HBDSP may promote the HBV particles release through wt-p53-dependent hepatocellular apoptosis. Our findings suggest that blocking HBDSP-induced wt-p53-dependent apoptosis might have therapeutic values for chronic hepatitis B.

Association of clonal haematopoiesis with severe postoperative complications in patients undergoing radical oesophagectomy.

BACKGROUND: Clonal haematopoiesis (CH) is an age-associated clonal expansion of blood cells driven by leukaemia-associated somatic mutations. Although CH has been reported to be a risk factor for leukaemia and a number of non-haematopoietic diseases, its role in perioperative medicine remains unexplored. METHODS: This was a single-centre, prospective, observational study. Patients undergoing radical oesophagectomy were enrolled, and peripheral blood samples were collected for DNA sequencing. Patients with haematopoietic somatic mutations (variant allele frequencies ≥1%) in the DNMT3A gene, TET2 gene, or both were defined as CH carriers. The primary outcome was the incidence of severe postoperative complications (Clavien-Dindo classification ≥3). The secondary outcomes included the major types of postoperative complications, mortality, and other common perioperative variables. RESULTS: Clonal haematopoiesis was found in 21.2% (33/156) of the patients (mean age: 66 yr [range: 26-79 yr]; 83% males). Some 14/33 (42.4%) patients with CH had severe postoperative complications, compared with patients without CH carriers (28/123 [22.8%]; P=0.024). Multivariable logistic regression analysis showed that CH was associated with an increased risk of developing severe postoperative complications (odds ratio, 3.63; 95% confidence interval, 1.37-9.66; P=0.010). Among the major postoperative complications, the incidence of pulmonary complications was significantly higher in the patients with CH than in those without CH (15 in 33 [45.5%] vs 30 in 123 [24.4%], P=0.018). CONCLUSIONS: Clonal haematopoiesis was associated with a higher incidence of severe postoperative complications in patients undergoing radical oesophagectomy, suggesting that clonal haematopoiesis can play an important role in perioperative medicine. CLINICAL TRIAL REGISTRATION: ChiCTR2100044175 (Chinese Clinical Trial Registry, http://www.chictr.org.cn/showproj.aspx?proj=123193).

Effect of clock rhythm on emergence agitation and early postoperative delirium in older adults undergoing thoracoscopic lung cancer surgery: protocol for a prospective, observational, cohort study.

INTRODUCTION: Surgeries conducted at night can impact patients' prognosis, and the mechanism may be related to circadian rhythm, which influence normal physiological functions and pathophysiological changes. Melatonin is primarily a circadian hormone with hypnotic and chronobiotic effects, thereby affecting disease outcomes through influencing the expression of inflammatory factors and biochemical metabolism. This study aims to observe the effects of circadian rhythms on emergence agitation and early postoperative delirium of older individuals undergoing thoracoscopic lung cancer surgery and explore the possible regulatory role of melatonin. METHODS: This prospective, observational, cohort study will involve 240 patients. Patients will be routinely divided into three groups based on the time of the surgery: T1 (8:00-14:00), T2 (14:00-20:00) and T3 group (20:00-08:00). The primary outcome will be the incidence of emergence agitation assessed via the Richmond Agitation and Sedation Scale (RASS) in the post-anesthesia care unit (PACU). Secondary outcomes will include the incidence of early postoperative delirium assessed via the Confusion Assessment Method (CAM) on postoperative day 1, pain status assessed via the numerical rating scale (NRS) in the PACU, sleep quality on postoperative day 1 and changes in perioperative plasma melatonin, clock genes and inflammatory factor levels. Postoperative surgical complications, intensive care unit admission and hospital length of stay will also be evaluated. DISCUSSION: This paper describes a protocol for investigating the effects of circadian rhythms on emergence agitation and early postoperative delirium of older individuals undergoing thoracoscopic lung cancer surgery, as well as exploring the potential regulatory role of melatonin. By elucidating the mechanism by which circadian rhythms impact postoperative recovery, we aim to develop a new approach for achieving rapid recovery during perioperative period. TRIAL REGISTRATION: The study was registered at the Chinese Clinical Trials Registry (ChiCTR2000040252) on November 26, 2020, and refreshed on September 4, 2022.

Hepatitis B Virus X Protein Is Stabilized by the Deubiquitinating Enzyme VCPIP1 in a Ubiquitin-Independent Manner by Recruiting the 26S Proteasome Subunit PSMC3.

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, and the viral X protein (HBx) is an etiological factor in HCC development. HBx is a high-turnover protein, but knowledge of the role of deubiquitinating enzymes (DUBs) in maintaining HBx homeostasis is very limited. We used a 74-DUB library-based yeast two-hybrid assay and determined that a novel DUB, valosin-containing protein-interacting protein 1 (VCPIP1), interacted with HBx. VCPIP1 and its C-terminal amino acids 863 to 1221 upregulated the HBx protein expression, with or without HBV infection. Mechanistically, VCPIP1 stabilized HBx protein through a ubiquitin-independent pathway, which was validated by the HBx ubiquitination site mutant plasmid. Coimmunoprecipitation assays demonstrated the potency of VCPIP1 in recruiting 26S proteasome regulatory subunit 6A (PSMC3) and forming a ternary complex with HBx through mutual interaction. In vitro, purified His-tagged PSMC3 protein rescued HBx degradation induced by the 20S proteasome, and in vivo VCPIP1 synergized the mechanism. Functionally, HBx specifically binding to VCPIP1 significantly enhanced the transcriptional transactivation of HBx by activating NF-κB, AP-1, and SP-1 and inhibited hepatoma cell clonogenicity in Huh7 and HepG2 cells. Moreover, we further demonstrated that overexpression of VCPIP1 significantly affected the HBV covalently closed circular DNA (cccDNA) transcription in HBV-infected HepG2-NTCP cells. Altogether, our results indicate a novel mechanism by which VCPIP1 recruits PSMC3 to bind with HBx, stabilizing it in a ubiquitin-independent manner, which might be critical for developing DUB inhibitors in the future. IMPORTANCE HBx is a multifunctional viral oncoprotein that plays an essential role in the viral life cycle and hepatocarcinogenesis. HBx degradation occurs through the ubiquitin-proteasome system (UPS). However, whether novel compartments of the DUBs in the UPS also act in regulating HBx stability is not fully understood. Here, for the first time, we defined VCPIP1 as a novel DUB for preventing HBx degradation by the 20S proteasome in a ubiquitin-independent manner. PSMC3, encoding the 26S proteasome regulatory subunit, directly stabilized HBx through physical binding instead of a common approach in protein degradation, serving as the key downstream effector of VCPIP1 on HBx. Therefore, the ternary binding pattern between VCPIP1, HBx, and PSMC3 is initiated for the first time, which eventually promotes HBx stability and its functions. Our findings provide novel insights into host-virus cross talk by targeting DUBs in the UPS.

Opioid-Free Versus Opioid-Based Anesthesia on Postoperative Pain After Thoracoscopic Surgery: The Use of Intravenous and Epidural Esketamine.

BACKGROUND: The effectiveness and safety of opioid-free anesthesia (OFA) regimens in distinct types of surgeries remain controversial. In this study, we investigated whether OFA could reduce the occurrence of chronic postoperative pain in patients receiving video-assisted thoracoscopic surgery (VATS). METHODS: We conducted a 2-center, randomized, controlled trial from September 2021 to January 2022. A total of 162 lung tumor patients scheduled to undergo VATS were randomly divided into an opioid-based anesthesia (OA) group and an OFA group. The OA group received general anesthesia combined with thoracic epidural block using morphine, while the OFA group received general anesthesia combined with thoracic epidural block using esketamine. Patient-controlled epidural analgesia (PCEA) was used after surgery (ropivacaine and morphine for the OA group versus ropivacaine and esketamine for the OFA group). The primary end point was chronic pain rates at 3 months after VATS, which were analyzed using a logistic regression model. The secondary end points were chronic pain rates at 6 months, acute pain rates at 24 hours and 48 hours postoperatively, postoperative side effects, and perioperative variables. RESULTS: The final analysis included 159 patients. Acute postoperative pain at 24 hours occurred in 0 of the 79 (0%) patients in the OA group and 10 of the 80 (17.5%) patients in the OFA group (odds ratio, 52.14; 95% confidence interval [CI], 6.47-420.10; P < .001). Acute postoperative pain at 48 hours occurred in 3 of the 79 (3.8%) patients in the OA group and 2 of the 80 (2.5%) patients in the OFA group (odds ratio, 2.07; 95% CI, 0.99-4.32; P = .053). In this study, none of the patients had moderate or severe pain in either group at 3 and 6 months postsurgically. Mild chronic postoperative pain at 3 months occurred in 27 of the 79 (34.2%) patients in the OA group and 14 of the 80 (17.5%) patients in the OFA group (odds ratio, 3.52; 95% CI, 1.49-8.31; P = .004). At 6 months, mild chronic pain still occurred in 23 of the 79 (29.1%) patients in the OA group and 9 of the 80 (11.3%) patients in the OFA group (odds ratio, 5.55; 95% CI, 2.01-15.33; P = .001). In addition, the OFA group included fewer patients with side effects, including nausea, vomiting, and pruritus, within 48 hours after surgery. CONCLUSIONS: Replacement of opioids by esketamine, intraoperatively as intravenous injection and epidural infusion and postoperatively as epidural infusion, reduces the incidence of mild chronic postoperative pain and side effects in patients after VATS.

Therapeutic Targeting of Tumor Cells Rich in LGR Stem Cell Receptors.

LGR5 and LGR6 mark epithelial stem cells in many niches including the ovarian surface and fallopian tube epithelia from which ovarian cancer arises. Human ovarian cancers express these receptors at high levels and express one of their ligands, RSPO1, at levels uniquely higher than all other tumor types except mesothelioma. Reasoning that these receptors are also important to tumor stem cells, arming the LGR binding domain of RSPO1 with a cytotoxin may permit depletion of the tumor stem cells. The Fu1-Fu2 receptor binding domain of RSPO1 (R1FF), containing a sortase recognition sequence at the C-terminal end, was produced in bacteria and a single molecule of MMAE was attached to each R1FF through a val-cit-PAB linker using the sortase reaction, thus producing a homogeneous population of armed molecules. R1FF-MMAE demonstrated (1) selective LGR-dependent binding, uptake, and cytotoxicity; (2) low nM cytotoxicity to multiple types of human tumor cell lines in vitro; (3) favorable plasma pharmacokinetic properties when administered iv with an elimination half-life of 27.8 h; (4) favorable absorption from the peritoneal cavity; and (5) therapeutic activity in aggressive xenograft models of ovarian cancer in the absence of any weight loss or other adverse events. These results demonstrate that the Fu1-Fu2 domain of RSPO1 can be exploited to deliver a potent cytotoxin to tumor cells that express the LGR4-6 family of stem cell receptors.

Hepatitis B Virus Surface Antigen Enhances the Sensitivity of Hepatocytes to Fas-Mediated Apoptosis via Suppression of AKT Phosphorylation.

The Fas receptor/ligand system plays a prominent role in hepatic apoptosis and hepatocyte death. Although hepatitis B virus (HBV) surface Ag (HBsAg) is the most abundant HBV protein in the liver and peripheral blood of patients with chronic HBV infection, its role in Fas-mediated hepatocyte apoptosis has not been disclosed. In this study, we report that HBsAg sensitizes HepG2 cells to agonistic anti-Fas Ab CH11-induced apoptosis through increasing the formation of SDS-stable Fas aggregation and procaspase-8 cleavage but decreasing both the expression of cellular FLIPL/S and the recruitment of FLIPL/S at the death-inducing signaling complex (DISC). Notably, HBsAg increased endoplasmic reticulum stress and consequently reduced AKT phosphorylation by deactivation of phosphoinositide-dependent kinase-1 (PDPK1) and mechanistic target of rapamycin complex 2 (mTORC2), leading to enhancement of Fas-mediated apoptosis. In a mouse model, expression of HBsAg in mice injected with recombinant adenovirus-associated virus 8 aggravated Jo2-induced acute liver failure, which could be effectively attenuated by the AKT activator SC79. Based on these results, it is concluded that HBsAg predisposes hepatocytes to Fas-mediated apoptosis and mice to acute liver failure via suppression of AKT prosurviving activity, suggesting that interventions directed at enhancing the activation or functional activity of AKT may be of therapeutic value in Fas-mediated progressive liver cell injury and liver diseases.

AKT activator SC79 protects hepatocytes from TNF-α-mediated apoptosis and alleviates d-Gal/LPS-induced liver injury.

Tumor necrosis factor-α (TNF-α) is a highly pleiotropic cytokine executing biological functions as diverse as cell proliferation, metabolic activation, inflammatory responses, and cell death. TNF-α can induce multiple mechanisms to initiate apoptosis in hepatocytes leading to the subsequent liver injury. Since the phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathway is known to have a protective role in death factor-mediated apoptosis, it is our hypothesis that activation of Akt may represent a therapeutic strategy to alleviate TNF-α-induced hepatocyte apoptosis and liver injury. We report here that the Akt activator SC79 protects hepatocytes from TNF-α-induced apoptosis and protects mice from d-galactosamine (d-Gal)/lipopolysaccharide (LPS)-induced TNF-α-mediated liver injury and damage. SC79 not only enhances the nuclear factor-κB (NF-κB) prosurvival signaling in response to TNF-α stimulation, but also increases the expression of cellular FLICE (FADD-like IL-1ß-converting enzyme)-inhibitory protein L and S (FLIPL/S), which consequently inhibits the activation of procaspase-8. Furthermore, pretreatment of the PI3K/Akt inhibitor LY294002 reverses all the SC79-induced hepatoprotective effects. These results strongly indicate that SC79 protects against TNF-α-induced hepatocyte apoptosis and suggests that SC79 is likely a promising therapeutic agent for ameliorating the development of liver injury. NEW & NOTEWORTHY SC79 protects hepatocytes from TNF-α-mediated apoptosis and mice from Gal/LPS-induced liver injury and damage. Cytoprotective effects of SC79 against TNF-α act through both AKT-mediated activation of NF-κB and upregulation of FLIPL/S.

Hepatitis B Spliced Protein (HBSP) Suppresses Fas-Mediated Hepatocyte Apoptosis via Activation of PI3K/Akt Signaling.

Hepatitis B spliced protein (HBSP) is known to associate with viral persistence and pathogenesis; however, its biological and clinical significance remains poorly defined. Acquired resistance to Fas-mediated apoptosis is thought to be one of the major promotors for hepatitis B virus (HBV) chronicity and malignancy. The purpose of this study was to investigate whether HBSP could protect hepatocytes against Fas-initiated apoptosis. We showed here that HBSP mediated resistance of hepatoma cells or primary human hepatocytes (PHH) to agonistic anti-Fas antibody (CH11)- or FasL-induced apoptosis. Under Fas signaling stimulation, expression of HBSP inhibited Fas aggregation and prevented recruitment of the adaptor molecule Fas-associated death domain (FADD) and procaspase-8 (or FADD-like interleukin-1ß-converting enzyme [FLICE]) into the death-inducing signaling complex (DISC) while increasing recruitment of cellular FLICE-inhibitory protein L (FLIPL) into the DISC. Those effects may be mediated through activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway as evidenced by increased cellular phosphatidylinositol (3,4,5)-trisphosphate (PIP3) content and PI3K activity and enhanced phosphorylation of mTORC2 and PDPK1 as well as Akt itself. Confirmedly, inhibition of PI3K by LY294002 reversed the effect of HBSP on Fas aggregation, FLIPL expression, and cellular apoptosis. These results indicate that HBSP functions to prevent hepatocytes from Fas-induced apoptosis by enhancing PI3K/Akt activity, which may contribute to the survival and persistence of infected hepatocytes during chronic infection.IMPORTANCE Our study revealed a previously unappreciated role of HBSP in Fas-mediated apoptosis. The antiapoptotic activity of HBSP is important for understanding hepatitis B virus pathogenesis. In particular, HBV variants associated with hepatoma carcinoma may downregulate apoptosis of hepatocytes through enhanced HBSP expression. Our study also found that Akt is centrally involved in Fas-induced hepatocyte apoptosis and revealed that interventions directed at inhibiting the activation or functional activity of Akt may be of therapeutic value in this process.

A Novel AKT Activator, SC79, Prevents Acute Hepatic Failure Induced by Fas-Mediated Apoptosis of Hepatocytes.

Acute liver failure is a serious clinical problem of which the underlying pathogenesis remains unclear and for which effective therapies are lacking. The Fas receptor/ligand system, which is negatively regulated by AKT, is known to play a prominent role in hepatocytic cell death. We hypothesized that AKT activation may represent a strategy to alleviate Fas-induced fulminant liver failure. We report here that a novel AKT activator, SC79, protects hepatocytes from apoptosis induced by agonistic anti-Fas antibody CH11 (for humans) or Jo2 (for mice) and significantly prolongs the survival of mice given a lethal dose of Jo2. Under Fas-signaling stimulation, SC79 inhibited Fas aggregation, prevented the recruitment of the adaptor molecule Fas-associated death domain (FADD) and procaspase-8 [or FADD-like IL-1ß-converting enzyme (FLICE)] into the death-inducing signaling complex (DISC), but SC79 enhanced the recruitment of the long and short isoforms of cellular FLICE-inhibitory protein at the DISC. All of the SC79-induced hepatoprotective and DISC-interruptive effects were confirmed to have been reversed by the Akt inhibitor LY294002. These results strongly indicate that SC79 protects hepatocytes from Fas-induced fatal hepatic apoptosis. The potent alleviation of Fas-mediated hepatotoxicity by the relatively safe drug SC79 highlights the potential of our findings for immediate hepatoprotective translation.

PI3K/AKT inhibitors aggravate death receptor-mediated hepatocyte apoptosis and liver injury.

The PI3K/AKT signaling pathway is one of the most frequently activated signaling networks in human cancers and has become a valuable target in anticancer therapy. However, accumulating reports suggest that adverse effects such as severe liver injury and inflammation may accompany treatment with pan-PI3K and pan-AKT inhibitors. Our prior work has demonstrated that activation of the PI3K/AKT pathway has a protective role in Fas- or TNFα-induced hepatocytic cell death and liver injury. We postulated that PI3K or AKT inhibitors may exacerbate liver damage via the death factor-mediated hepatocyte apoptosis. In this study we found that several drugs targeting PI3K/AKT either clinically used or in clinical trials sensitized hepatocytes to agonistic anti-Fas antibody- or TNFα-induced apoptosis and significantly shortened the survival of mice in in vivo liver damage models. The PI3K or AKT inhibitors promoted Fas aggregation, inhibited the expression of cellular FLICE-inhibitory protein S and L (FLIPL/S), and enhanced procaspase-8 activation. Conversely, cotreatment with the AKT specific activator SC79 reversed these effects. Taken together, these findings suggest that PI3K or AKT inhibitors may render hepatocytes hypersensitive to Fas- or TNFα-induced apoptosis and liver injury.

Aberrantly expressed miR-188-5p promotes gastric cancer metastasis by activating Wnt/ß-catenin signaling.

BACKGROUND: Gastric cancer (GC) is one of the most common human cancers with the high rate of recurrence, metastasis and mortality. Aberrantly expressed microRNAs (miRNAs) are associated with invasion and metastasis in various human cancers. Recently, miR-188-5p has been indicated as an oncogene in GC since it promotes GC cell growth and metastasis. However, the underlying molecular mechanism remains to be fully defined. METHODS: Using Significance Analysis of Microarrays (SAM) screening, we identified that miR-188-5p is associated with overall survival and lymph node metastasis in patients with GC. The functional impact of miR-188-5p on GC metastasis was validated using in vitro and in vivo assays. The regulatory function of miR-188-5p on Wnt/ß-catenin signaling activation through directly targeting PTEN was proven using quantitative real-time PCR, western blot analysis, a dual-luciferase assay, a Transwell assay, and immunofluorescence. Immunohistochemical analyses further confirmed the clinical significance of miR-188-5p in GC. RESULTS: MiR-188-5p diminishes tumor suppressor PTEN expression, and further increases phospho-Ser9 of GSK3ß to activate Wnt/ß-catenin signaling in GC. Consequently, miR-188-5p enhanced the migration and invasion of GC cells in vitro and tumor metastasis in vivo, whereas inhibition of miR-188-5p had the opposite effects. Moreover, miR-188-5p was negatively correlated with PTEN expression but positively correlated with nuclear ß-catenin staining in GC samples. CONCLUSIONS: Our findings revealed a model of the miR-188-5p-PTEN-ß-catenin axis in GC, which mediates the constitutive activation of Wnt/ß-catenin signaling and promotes tumor metastasis, inferring that miR-188-5p is a potential therapeutic target to treat GC.

Hepatitis B Virus X Protein Increases 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine (8-Oxodg) Level via Repressing MTH1/ MTH2 Expression in Hepatocytes.

BACKGROUND/AIMS: Chronic hepatitis B virus (HBV) infection markedly increases the risk of development of hepatocellular carcinoma (HCC). Among the seven viral proteins that HBV encodes, HBV X protein (HBx) appears to have the most oncogenic potential. The mitochondria-associated HBx can induce oxidative stress in hepatocytes, leading to the production of abundant reactive oxygen species (ROS). High levels of ROS usually induce oxidative DNA damage and 8-hydroxy-2-deoxyguanosine (8-OHdG), also known as 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), which is one of the major products of DNA oxidation and an important biomarker for oxidative stress and carcinogenesis. Cells have evolved a mechanism to prevent oxidized nucleotides from their incorporation into DNA through nucleotide pool sanitization enzymes of MTH1 (NUDT1), MTH2 (NUDT15), MTH3 (NUDT18) and NUDT5. However, little is known as to whether HBx can regulate the expression of those enzymes and modulate the formation and accumulation of 8-oxodG in hepatocytes. METHODS: The level of 8-oxodG was assessed by ELISA in stable HBV-producing hepatoma cell lines, an HBV infectious mouse model, HBV and HBx transgenic mice and HBV-infected patients versus their respective controls. Expression of MTH1, MTH2, MTH3 and NUDT5 was determined by a real-time quantitative PCR and western blot analysis. Transcriptional regulation of MTH1 and MTH2 expression by HBx and the effect of HBx on MTH1 and MTH2 promoter hypermethylation were examined using a luciferase reporter assay and bisulfite sequencing analysis. RESULTS: In comparison with controls, significantly higher levels of 8-oxodG were detected in the genome and culture supernatant of stable HBV-producing HepG2.2.15 cells, in the sera and liver tissues of HBV infectious mice and HBV or HBx transgenic mice, and in the sera of HBV-infected patients. Expression of HBx in hepatocytes significantly increased 8-oxodG level and reduced the expression of MTH1 and MTH2 at both mRNA and protein levels. It was also demonstrated that HBx markedly attenuated the MTH1 or MTH2 promoter activities through hypermethylation. Furthermore, enhancement of 8-oxodG production by HBx was reversible by overexpression of MTH1 and MTH2. CONCLUSION: Our data show that HBx expression results in the accumulation of 8-oxodG in hepatocytes through inhibiting the expression of MTH1 and MTH2. This may implicate that HBx may act as a tumor promoter through facilitating the mutational potential of 8-oxodG thus connecting a possible link between HBV infection and liver carcinogenesis.

Hepatitis B Virus X Protein Induces Hepatic Steatosis by Enhancing the Expression of Liver Fatty Acid Binding Protein.

UNLABELLED: Hepatitis B virus (HBV) has been implicated as a potential trigger of hepatic steatosis although molecular mechanisms involved in the pathogenesis of HBV-associated hepatic steatosis still remain elusive. Our prior work has revealed that the expression level of liver fatty acid binding protein 1 (FABP1), a key regulator of hepatic lipid metabolism, was elevated in HBV-producing hepatoma cells. In this study, the effects of HBV X protein (HBx) mediated FABP1 regulation on hepatic steatosis and the underlying mechanism were determined. mRNA and protein levels of FABP1 were measured by quantitative RT-PCR (qPCR) and Western blotting. HBx-mediated FABP1 regulation was evaluated by luciferase assay, coimmunoprecipitation, and chromatin immunoprecipitation. Hepatic lipid accumulation was measured by using Oil-Red-O staining and the triglyceride level. It was found that expression of FABP1 was increased in HBV-producing hepatoma cells, the sera of HBV-infected patients, and the sera and liver tissues of HBV-transgenic mice. Ectopic overexpression of HBx resulted in upregulation of FABP1 in HBx-expressing hepatoma cells, whereas HBx abolishment reduced FABP1 expression. Mechanistically, HBx activated the FABP1 promoter in an HNF3ß-, C/EBPα-, and PPARα-dependent manner, in which HBx increased the gene expression of HNF3ß and physically interacted with C/EBPα and PPARα. On the other hand, knockdown of FABP1 remarkably blocked lipid accumulation both in long-chain free fatty acids treated HBx-expressing HepG2 cells and in a high-fat diet-fed HBx-transgenic mice. Therefore, FABP1 is a key driver gene in HBx-induced hepatic lipid accumulation via regulation of HNF3ß, C/EBPα, and PPARα. FABP1 may represent a novel target for treatment of HBV-associated hepatic steatosis. IMPORTANCE: Accumulating evidence from epidemiological and experimental studies has indicated that chronic HBV infection is associated with hepatic steatosis. However, the molecular mechanism underlying HBV-induced pathogenesis of hepatic steatosis still remains to be elucidated. In this study, we found that expression of liver fatty acid binding protein (FABP1) was dramatically increased in the sera of HBV-infected patients and in both sera and liver tissues of HBV-transgenic mice. Forced expression of HBx led to FABP1 upregulation, whereas knockdown of FABP1 remarkably diminished lipid accumulation in both in vitro and in vivo models. It is possible that HBx promotes hepatic lipid accumulation through upregulating FABP1 in the development of HBV-induced nonalcoholic fatty liver disease. Therefore, inhibition of FABP1 might have therapeutic value in steatosis-associated chronic HBV infection.

Hepatitis B virus core protein inhibits Fas-mediated apoptosis of hepatoma cells via regulation of mFas/FasL and sFas expression.

Hepatitis B virus core protein (HBc) has been implicated in hepatocarcinogenesis through several mechanisms. Resistance of hepatitis B virus (HBV)-infected hepatocytes to apoptosis is considered one of the major contributors to the progression of chronic hepatitis to cirrhosis and ultimately to hepatocellular carcinoma. The Fas receptor/ligand (Fas/FasL) system plays a prominent role in hepatocyte death during HBV infection. Here we report that HBc mediates resistance of hepatoma cells to agonistic anti-Fas antibody (CH11)-induced apoptosis. When HBc was introduced into human hepatoma cells, the cells became resistant to CH11 cytotoxicity in a p53-dependent manner. HBc significantly down-regulated the expression of p53, total Fas, and membrane-bound Fas at the mRNA and protein levels and reduced FasL mRNA expression. In contrast, HBc up-regulated the expression of soluble forms of Fas by increasing Fas alternative mRNA splicing. Mechanistically, HBc-mediated Fas alternative mRNA splicing was associated with up-regulation of polypyrimidine tract-binding protein 1 and down-regulation of Fas-activated serine/threonine kinase. These results indicated that HBc may prevent hepatocytes from Fas-induced apoptosis by the dual effects of reducing the expression of the proapoptotic form of Fas and enhancing the expression of the antiapoptotic form of the receptor, which may contribute to the survival and persistence of infected hepatocytes during chronic infection.

Hepatitis B spliced protein (HBSP) promotes the carcinogenic effects of benzo [alpha] pyrene by interacting with microsomal epoxide hydrolase and enhancing its hydrolysis activity.

BACKGROUND: The risk of hepatocellular carcinoma (HCC) increases in chronic hepatitis B surface antigen (HBsAg) carriers who often have concomitant increase in the levels of benzo[alpha]pyrene-7,8-diol-9,10-epoxide(±) (BPDE)-DNA adduct in liver tissues, suggesting a possible co-carcinogenesis of Hepatitis B virus (HBV) and benzo[alpha]pyrene in HCC; however the exact mechanisms involved are unclear. METHODS: The interaction between hepatitis B spliced protein (HBSP) and microsomal epoxide hydrolase (mEH) was confirmed using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assay; the effects of HBSP on mEH-mediated B[alpha]P metabolism was examined by high performance liquid chromatography (HPLC); and the influences of HBSP on B[alpha]P carcinogenicity were evaluated by bromodeoxyuridine cell proliferation, anchorage-independent growth and tumor xenograft. RESULTS: HBSP could interact with mEH in vitro and in vivo, and this interaction was mediated by the N terminal 47 amino acid residues of HBSP. HBSP could greatly enhance the hydrolysis activity of mEH in cell-free mouse liver microsomes, thus accelerating the metabolism of benzo[alpha]pyrene to produce more ultimate carcinnogen, BPDE, and this effect of HBSP requires the intact HBSP molecule. Expression of HBSP significantly increased the formation of BPDE-DNA adduct in benzo[alpha]pyrene-treated Huh-7 hepatoma cells, and this enhancement was blocked by knockdown of mEH. HBSP could enhance the cell proliferation, accelerate the G1/S transition, and promote cell transformation and tumorigenesis of B[alpha]P-treated Huh-7 hepatoma cells. CONCLUSIONS: Our results demonstrated that HBSP could promote carcinogenic effects of B[alpha]P by interacting with mEH and enhancing its hydrolysis activity.

Effect of music on hemodynamic fluctuations in women during induction of general anesthesia: A prospective randomized controlled multicenter trial.

BACKGROUND: The authors aim to investigate the effect of music on hemodynamic fluctuations during induction of general anesthesia and reducing preoperative anxiety for women who underwent elective non-cardiac surgery. METHODS: It is a multicenter, double-blind, randomized, parallel-group clinical trial. Patients were randomized 1:1 to either a Music Intervention group (MI) or a Control group (Control). The MI participants listened to their preferred music for more than 30 minutes in the waiting area. The State-Trait Anxiety Inventory (STAI) was used to measure anxiety levels in the groups, and hemodynamic parameters (Heart Rate [HR], Mean Arterial Pressure [MAP]) were continuously recorded before induction (T0), at loss of consciousness (T1), immediately before intubation (T2), and after intubation (T3). Intubation-related adverse events were also recorded. The primary outcome was the incidence of MAP changes more than 20 % above baseline during T0-T2. RESULTS: A total of 164 patients were included in the final analyses. The incidence of MAP instability during T0-T2 was lower in the MI, and the 95 % Confidence Interval for the rate difference demonstrated the superiority of MI. HR instability was less frequent in MI participants both in T0-T2 and T2-T3. The overall incidence of preoperative anxiety was 53.7 % (88/164). After the music intervention, the mean score of STAI was significantly lower in the MI than in the Control, with a between-group difference of 8.01. CONCLUSIONS: Preoperative music intervention effectively prevented hemodynamic instability during anesthesia induction and significantly reduced preoperative anxiety in women undergoing elective non-cardiac surgery.

Exploring biomarkers for diagnosing and predicting organ dysfunction in patients with perioperative sepsis: a preliminary investigation.

OBJECTIVE: Early diagnosis and prediction of organ dysfunction are critical for intervening and improving the outcomes of septic patients. The study aimed to find novel diagnostic and predictive biomarkers of organ dysfunction for perioperative septic patients. METHOD: This is a prospective, controlled, preliminary, and single-center study of emergency surgery patients. Mass spectrometry, Gene Ontology (GO) functional analysis, and the protein-protein interaction (PPI) network were performed to identify the differentially expressed proteins (DEPs) from sepsis patients, which were selected for further verification via enzyme-linked immunosorbent assay (ELISA). Logistic regression analysis was used to estimate the relative correlation of selected serum protein levels and clinical outcomes of septic patients. Calibration curves were plotted to assess the calibration of the models. RESULTS: Five randomized serum samples per group were analyzed via mass spectrometry, and 146 DEPs were identified. GO functional analysis and the PPI network were performed to evaluate the molecular mechanisms of the DEPs. Six DEPs were selected for further verification via ELISA. Cathepsin B (CatB), vascular cell adhesion protein 1 (VCAM-1), neutrophil gelatinase-associated lipocalin (NGAL), protein S100-A9, prosaposin, and thrombospondin-1 levels were significantly increased in the patients with sepsis compared with those of the controls (p < 0.001). Logistic regression analysis showed that CatB, S100-A9, VCAM-1, prosaposin, and NGAL could be used for preoperative diagnosis and postoperative prediction of organ dysfunction. CatB and S100-A9 were possible predictive factors for preoperative diagnosis of renal failure in septic patients. Internal validation was assessed using the bootstrapping validation. The preoperative diagnosis of renal failure model displayed good discrimination with a C-index of 0.898 (95% confidence interval 0.843-0.954) and good calibration. CONCLUSION: Serum CatB, S100-A9, VCAM-1, prosaposin, and NGAL may be novel markers for preoperative diagnosis and postoperative prediction of organ dysfunction. Specifically, S100-A9 and CatB were indicators of preoperative renal dysfunction in septic patients. Combining these two biomarkers may improve the accuracy of predicting preoperative septic renal dysfunction. TRIAL REGISTRATION: The study was registered at the Chinese Clinical Trials Registry (ChiCTR2200060418) on June 1, 2022.

Interaction of the hepatitis B spliced protein with cathepsin B promotes hepatoma cell migration and invasion.

Hepatitis B spliced protein (HBSP) is involved in the pathogenicity and/or persistence of hepatitis B virus (HBV). Chronic HBV infection is one of the most important risk factors for the development of hepatocellular carcinoma (HCC). However, whether or not HBSP contributes to the progression of HBV-associated HCC remains unknown. This study reports that overexpression of HBSP in human hepatoma cells increased cell invasion and motility. Conversely, small interfering RNA (siRNA)-mediated knockdown of HBSP expression inhibited migration and invasion. By glutathione S-transferase (GST) pulldown, coimmunoprecipitation, and a mammalian two-hybrid assay, HBSP was found to directly interact with cathepsin B (CTSB). Similar to HBSP knockdown, knocking down CTSB also reduced cell migration and invasion. Furthermore, the HBSP-overexpressing hepatoma cells were shown to have increased expression and activity of matrix metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator (uPA), and overexpression of HBSP significantly enhanced tumor-induced vascularization of endothelial cells. In contrast, knockdown of either HBSP or CTSB by siRNA resulted in inhibition of the two proteolytic enzymes and of the in vitro angiogenesis. Expression of HBSP in the hepatoma cells appeared to activate the mitogen-activated protein kinase (MAPK) and Akt signaling pathway, as evidenced by increases in phosphorylation of p38, Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt. Taken together, these findings imply that interaction of HBSP with CTSB may promote hepatoma cell motility and invasion and highlight new molecular mechanisms for HBSP-induced HCC progression that involve the secretion and activation of proteolytic enzymes, increased tumor-induced angiogenesis, and activation of the MAPK/Akt signaling, thereby leading to the aggressiveness of hepatoma cells.