Bicyclol ameliorates advanced liver diseases in murine models via inhibiting the IL-6/STAT3 signaling pathway.

Bicyclol, a synthetic hepatoprotective and anti-inflammatory agent approved in China, was widely used to treat various hepatitis accompanied by elevated serum aminotransferases. However, the pharmacological effects and mechanisms of bicyclol on advanced liver diseases, such as fibrosis/cirrhosis and hepatocellular carcinoma (HCC), remain to be explored. Here, we revealed that bicyclol prevents from formatting severe fibrosis, slows the progression of moderate liver fibrosis, accelerates the regression of moderate liver fibrosis, decreases the malignancy of HCC in rat models induced by diethylnitrosamine (DEN), and also blocks steatohepatitis to HCC in mice induced by western diet plus carbon tetrachloride and DEN. The detailed pharmacological mechanism showed that bicyclol alleviates chronic progressive liver diseases by inhibiting the levels of IL-6 and subsequent phosphorylated STAT3. Conclusion: Bicyclol plays significant protective roles in multiply stages of fibrosis/cirrhosis-HCC and nonalcoholic fatty liver disease-related HCC via inhibiting IL-6/STAT3 signaling pathway. Therefore, bicyclol might be a promising therapeutic strategy for treating advanced liver diseases.

Combined Use of Bicyclol and Berberine Alleviates Mouse Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD), ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), is a liver disease worldwide without approved therapeutic drugs. Anti-inflammatory and hepatoprotective drug bicyclol and multi-pharmacological active drug berberine, respectively, have shown beneficial effects on NAFLD in murine nutritional models and patients, though the therapeutic mechanisms remain to be illustrated. Here, we investigated the combined effects of bicyclol and berberine on mouse steatosis induced by Western diet (WD), and NASH induced by WD/CCl4. The combined use of these was rather safe and better reduced the levels of transaminase in serum and triglycerides and cholesterol in the liver than their respective monotherapy, accompanied with more significantly attenuating hepatic inflammation, steatosis, and ballooning in mice with steatosis and NASH. The combined therapy also significantly inhibited fibrogenesis, characterized by the decreased hepatic collagen deposition and fibrotic surface. As per mechanism, bicyclol enhanced lipolysis and ß-oxidation through restoring the p62-Nrf2-CES2 signaling axis and p62-Nrf2-PPARα signaling axis, respectively, while berberine suppressed de novo lipogenesis through downregulating the expression of acetyl-CoA carboxylase and fatty acid synthetase, along with enrichment of lipid metabolism-related Bacteroidaceae (family) and Bacteroides (genus). Of note, the combined use of bicyclol and berberine did not influence each other but enhanced the overall therapeutic role in the amelioration of NAFLD. Conclusion: Combined use of bicyclol and berberine might be a new available strategy to treat NAFLD.

TGF-ß isoforms inhibit hepatitis C virus propagation in transforming growth factor beta/SMAD protein signalling pathway dependent and independent manners.

Transforming growth factor beta (TGF-ß) plays an important role in the viral liver disease progression via controlling viral propagation and mediating inflammation-associated responses. However, the antiviral activities and mechanisms of TGF-ß isoforms, including TGF-ß1, TGF-ß2 and TGF-ß3, remain unclear. Here, we demonstrated that all of the three TGF-ß isoforms were increased in Huh7.5 cells infected by hepatitis C virus (HCV), but in turn, the elevated TGF-ß isoforms could inhibit HCV propagation with different potency in infectious HCV cell culture system. TGF-ß isoforms suppressed HCV propagation through interrupting several different stages in the whole HCV life cycle, including virus entry and intracellular replication, in TGF-ß/SMAD signalling pathway-dependent and TGF-ß/SMAD signalling pathway-independent manners. TGF-ß isoforms showed additional anti-HCV activities when combined with each other. However, the elevated TGF-ß1 and TGF-ß2, not TGF-ß3, could also induce liver fibrosis with a high expression of type I collagen alpha-1 and α-smooth muscle actin in LX-2 cells. Our results showed a new insight into TGF-ß isoforms in the HCV-related liver disease progression.

Autophagy in fate determination of mesenchymal stem cells and bone remodeling.

Mesenchymal stem cells (MSCs) have been widely exploited as promising candidates in clinical settings for bone repair and regeneration in view of their self-renewal capacity and multipotentiality. However, little is known about the mechanisms underlying their fate determination, which would illustrate their effectiveness in regenerative medicine. Recent evidence has shed light on a fundamental biological role of autophagy in the maintenance of the regenerative capability of MSCs and bone homeostasis. Autophagy has been implicated in provoking an immediately available cytoprotective mechanism in MSCs against stress, while dysfunction of autophagy impairs the function of MSCs, leading to imbalances of bone remodeling and a wide range of aging and degenerative bone diseases. This review aims to summarize the up-to-date knowledge about the effects of autophagy on MSC fate determination and its role as a stress adaptation response. Meanwhile, we highlight autophagy as a dynamic process and a double-edged sword to account for some discrepancies in the current research. We also discuss the contribution of autophagy to the regulation of bone cells and bone remodeling and emphasize its potential involvement in bone disease.

Aloperine inhibits hepatitis C virus entry into cells by disturbing internalisation from endocytosis to the membrane fusion process.

Aloperine, a natural alkaloid isolated from the Chinese traditional herb Sophora alopecuroides, is a broad-spectrum antiviral agent with anti-inflammatory activity. Here, we found that aloperine effectively inhibited hepatitis C virus (HCV) propagation in Huh7.5 cells and primary human hepatocytes without cytotoxicity, and it blocked HCV cell-to-cell viral transmission. The antiviral mechanism evidence demonstrated that aloperine inhibits HCV internalisation from endocytosis to the membrane fusion process, and the target may be associated with host factors. Aloperine additively inhibited HCV propagation with direct-acting antivirals (DAAs) and was effective against HCV variants resistant to known DAAs. Therefore, aloperine might be a natural lead compound for the development of innovative antivirals, and the combined use of aloperine with DAAs might contribute to eliminating liver diseases caused by HCV infection.

A proof-of-concept study in HCV-infected Huh7.5 cells for shortening the duration of DAA-based triple treatment regimens.

With the development of more effective direct-acting antivirals (DAAs), dual- or triple-therapy regimens represent the major strategy used to cure chronic hepatitis C virus (HCV) infection. Thus, shorter treatment duration regimens with low burden, few adverse effects and good patient adherence are urgently needed. This study theoretically demonstrates a proof-of-concept approach for shortening therapy duration by examining HCV-infected Huh7.5 cells after treatment with a high or low fixed dose of three DAAs (simeprevir + daclatasvir + sofosbuvir) for 6-15 days. The results demonstrated that HCV-infected Huh7.5 cells achieved an ultrarapid virologic response with undetectable HCV RNA and protein and were cured after treatment with the triple-therapy regimen for 15 days. When the treatment duration was shortened, virologic relapse might occur after treatment with a low fixed dose of the three DAAs for 9 days and did occur after treatment with a low fixed dose for 6 days, although HCV was below detectable levels at the end of treatment. However, virologic relapse could be avoided with treatment of a high fixed dose of the three DAAs for 9 or 6 days. Although a virologic breakthrough occurred after an intermittent treatment regimen at the low fixed dose, the high fixed dose cured HCV-positive Huh7.5 cells with intermittent treatment. In conclusion, HCV is persistently present below detectable levels in HCV-infected Huh7.5 cells for a long time after treatment, and a shortened therapy duration is associated with an increased risk of virologic relapse, but virologic relapse or breakthrough might be avoided by treatment with a combination of more highly effective DAAs.

Bicyclol Attenuates Liver Inflammation Induced by Infection of Hepatitis C Virus via Repressing ROS-Mediated Activation of MAPK/NF-κB Signaling Pathway.

Treatment with direct-acting antivirals (DAAs) cures most patients infected with hepatitis C virus (HCV) in the real world. However, some patients, especially those with the underlying advanced liver disease, have a limited reduction of liver injury after achieving a sustained viral response (SVR). Bicyclol was widely used in clinics for the treatment of a variety of liver injuries but with an unknown mechanism for the treatment of hepatitis C. We investigated the anti-inflammatory effects and mechanisms of bicyclol in HCV-infected hepatocytes and further confirmed the putative results in a mouse hepatitis model induced by the coinjection of polyinosinic-polycytidylic acid [poly (I:C)] and D-galactosamine (D-GalN). The results showed that the activation of nuclear factor kappa B (NF-κB) and the subsequent increase of inflammatory factors were directly induced by HCV infection and were persistent after clearance of the virus in Huh7.5 cells. Bicyclol decreased the activation of NF-κB and the levels of inflammatory factors in HCV-infected hepatocytes by inhibiting the activation of the ROS-MAPK-NF-κB pathway, and the effect was synergistic with DAAs in HCV-infected hepatocytes. Bicyclol attenuated the ROS-MAPK-NF-κB axis via recovering mitochondrial function without a dependence on dihydronicotinamide adenine dinucleotide phosphate oxidase and superoxide dismutases. The anti-inflammatory effects and mechanism of bicyclol were verified in mouse hepatitis induced by the coinjection of poly(I:C)/D-GalN. Bicyclol directly ameliorates the chronic inflammation caused by HCV infection and might be used with DAAs or after DAA therapy for ultimately curing chronic hepatitis C.

Spatial signalling mediated by the transforming growth factor-ß signalling pathway during tooth formation.

Tooth development relies on sequential and reciprocal interactions between the epithelial and mesenchymal tissues, and it is continuously regulated by a variety of conserved and specific temporal-spatial signalling pathways. It is well known that suspensions of tooth germ cells can form tooth-like structures after losing the positional information provided by the epithelial and mesenchymal tissues. However, the particular stage in which the tooth germ cells start to form tooth-like structures after losing their positional information remains unclear. In this study, we investigated the reassociation of tooth germ cells suspension from different morphological stages during tooth development and the phosphorylation of Smad2/3 in this process. Four tooth morphological stages were designed in this study. The results showed that tooth germ cells formed odontogenic tissue at embryonic day (E) 14.5, which is referred to as the cap stage, and they formed tooth-like structures at E16.5, which is referred to as the early bell stage, and E18.5, which is referred to as the late bell stage. Moreover, the transforming growth factor-ß signalling pathway might play a role in this process.

[Orthodontic retention and adjustment of the occlusion after orthognathic surgery].

OBJECTIVE: To investigate the orthodontic retention and adjustment of the occlusion after orthognathic surgery. METHODS: 18 patients were divided into three groups. Group I: Cases with skeletal Class I bimaxillary protrusion treated by subapical osteotomy; Group II: Cases with skeletal Class II mandibular retrusion treated by sagittal split mandibular advancement surgery; Group III: Cases with skeletal Class III treated by Le Fort I osteotomy on maxilla and sagittal split osteotomy on mandible. There were 6 patients in each group. Three kinds of orthodontic elastic tractions were used based on different categories of malocclusion and different types of operation. RESULTS: 18 patients attained functional occlusion after the orthodontic occlusion adjustment. There was no relapse and malocclusion by surgery. CONCLUSION: Intermaxillary elastics based on different categories of occlusion and different kinds of surgery can improve the occlusion after orthognathic surgery and attatin the functional occlusion.

Transgenic mice for cre-inducible overexpression of the Cul4A gene.

The Cullin 4A(Cul4A) gene is important in cell survival, development, growth, and cell cycle control and is amplified in breast and hepatocellular cancers. Recently, we reported that Cul4A plays an oncogenic role in the pathogenesis of mesothelioma. An important strategy for studying Cul4A in different tissues is targeted overexpression of this gene in vivo. Studies of Cul4A in mice have been restricted to the loss-of-function studies using Cul4A knockout mice; gain-of-function studies of Cul4A using transgenic mice have not been reported. We, therefore, generated a gain-of-function transgenic mouse model that overexpresses Cul4A in a Cre-dependent manner. Before Cre recombination, these mice express LacZ during development in most adult tissues. After Cre-mediated excision of the LacZ reporter, the transfected Cul4A gene is expressed along with a C-terminal Myc-tag in different tissues. In this study, Cre-excision was induced in mouse lungs by inhalation of an adenovirus vector encoding Cre recombinase. This mouse model provides a valuable resource for investigating the significance of Cul4A overexpression in various tissues.

The expression of CXCR4, CXCL12 and CXCR7 in malignant pleural mesothelioma.

The chemokine CXCL12 and its receptors, CXCR4 and CXCR7, are involved in tumour progression, metastasis, and survival. We investigated the expression of CXCR4, CXCL12, and CXCR7 in malignant pleural mesothelioma to determine if they are possible biomarkers and potential therapeutic targets. Forty-one mesothelioma tumour tissues, ten normal human pleural tissues, and two mesothelioma cell lines were stained with anti-CXCR4, anti-CXCL12, anti-CXCR7, and anti-p-Akt antibodies. RT-PCR was performed to determine the expression of CXCR4, CXCL12, and CXCR7 in six human mesothelioma cell lines (H28, 211H, H2052, ms-1, H290, and H513) and one human normal mesothelial cell line, LP9. These seven cell lines were also stained with anti-CXCR7. We found that CXCR4 and CXCL12 were expressed in 97.6% and 78.0% mesothelioma tissue samples, concurrently with strong expression of p-Akt (R(2) = 0.739 and 0.620, respectively). In addition, CXCR7 expression was weaker than CXCR4 expression in mesothelioma tissues. Furthermore, RT-PCR showed that CXCR4 and CXCL12 were overexpressed in 5/6 mesothelioma cell lines (211H, H2052, ms-1, H290, and H513), whereas CXCR7 was overexpressed in only 2/6 (H513 and H2052). Moreover, we found that the CXCR4 antagonist AMD3100 inhibited the growth of all five mesothelioma cell lines that overexpress CXCR4 and CXCL12. Our results suggest that the Akt-mTOR pathway is involved during the interruption of the CXCL12/CXCR4 axis in these five mesothelioma cell lines. In conclusion, CXCR4 and CXCL12 are highly expressed in most mesothelioma cell lines and tumour tissues, suggesting that CXCR4 and CXCL12 may be used as biomarkers for patients with mesothelioma. The CXCL12-CXCR4 interaction may be a potential therapeutic target for mesothelioma.

Donor MHC gene to mitigate rejection of transplantation in recipient mice.

BACKGROUND: Donor organ rejection continues to be a significant problem for patients receiving transplants. We therefore tested whether transferring a donor's major histocompatibility complex (MHC) gene to the recipient would mitigate the rejection of transplanted hearts in mice. METHODS: H-2K(k) gene from donor mice was amplified using nested polymerase chain reaction (PCR) and ligated into a mammalian expression vector, which was then transfected into thymus ground mass cells collected from the recipients. Clones stably expressing the transgene were then injected into the recipients' thymus visualized using ultrasound. Control mice were administered cells previously transfected with empty vector. Following heart transplantation, cardiac activity was monitored electrocardiographically. Recipient thymus cells were tested for MHC antigenicity using flow cytometry and spleen cells were subjected to mixed lymphocyte culture tests. Finally, the transplanted hearts were sectioned, stained and examined under light microscopy. RESULTS: Southern analysis following nested PCR revealed clear expression of H-2K(k) gene. Following transplantation, electrocardiosignals were detectable highly significantly longer in recipients administered thymal cells expressing donor H-2K(k) than in those receiving control cells. Flow cytometric analysis using an anti-H-2K(k) antibody confirmed its expression in H-2K(k) treated recipients but not in control mice. Mixed lymphocyte cultures containing H-2K(k) treated cells showed significantly less proliferation than those containing control cells. Hearts from control mice showed substantially greater lymphocyte infiltration than those from H-2K(k) treated mice and large areas of necrosis. CONCLUSION: Rejection of transplanted hearts can be mitigated substantially by introducing the donor's MHC into the recipient.