Sevelamer reverses liver fibrosis by deactivation of hepatic stellate cells.

Liver fibrosis is a chronic liver disease characterized by a progressive wound healing response caused by chronic liver injury. Currently, there are no approved clinical treatments for liver fibrosis. Sevelamer is used clinically to treat hyperphosphatemia and has shown potential therapeutic effects on liver diseases. However, there have been few studies evaluating the therapeutic effects of sevelamer on liver fibrosis, and the specific mechanisms are still unclear. In this study, we investigated the antifibrotic effects of sevelamer-induced low inorganic phosphate (Pi) stress in vitro and in vivo and analyzed the detailed mechanisms. We found that low Pi stress could inhibit the proliferation of activated hepatic stellate cells (HSCs) by promoting apoptosis, effectively suppressing the migration and epithelial-mesenchymal transition (EMT) of hepatic stellate cells. Additionally, low Pi stress significantly increased the antioxidant stress response. It is worth noting that low Pi stress indirectly inhibited the activation and migration of HSCs by suppressing transforming growth factor ß (TGF-ß) expression in macrophages. In a rat model of liver fibrosis, oral administration of sevelamer significantly decreased blood phosphorus levels, improved liver function, reduced liver inflammation, and increased the antioxidant stress response in the liver. Our study revealed that the key mechanism by which sevelamer inhibited liver fibrosis involved binding to gastrointestinal phosphate, resulting in a decrease in blood phosphorus levels, the downregulation of TGF-ß expression in macrophages, and the inhibition of HSC migration and fibrosis-related protein expression. Therefore, our results suggest that sevelamer-induced low Pi stress can attenuate hepatic stellate cell activation and inhibit the progression of liver fibrosis, making it a potential option for the treatment of liver fibrosis and other refractory chronic liver diseases.

Irreversible repolarization of tumour-associated macrophages by low-Pi stress inhibits the progression of hepatocellular carcinoma.

Numerous studies have shown the positive correlation between high levels of Pi and tumour progression. A critical goal of macrophage-based cancer therapeutics is to reduce anti-inflammatory macrophages (M2) and increase proinflammatory antitumour macrophages (M1). This study aimed to investigate the relationship between macrophage polarization and low-Pi stress. First, the spatial populations of M2 and M1 macrophages in 22 HCC patient specimens were quantified and correlated with the local Pi concentration. The levels of M2 and M1 macrophage markers expressed in the peritumour area were higher than the intratumour levels, and the expression of M2 markers was positively correlated with Pi concentration. Next, monocytes differentiated from THP-1 cells were polarized against different Pi concentrations to investigate the activation or silencing of the expression of p65, IκB-α and STAT3 as well as their phosphorylation. Results showed that low-Pi stress irreversibly repolarizes tumour-associated macrophages (TAMs) towards the M1 phenotype by silencing stat6 and activating p65. Moreover, HepG-2 and SMCC-7721 cells were cultured in conditioned medium to investigate the innate anticancer immune effects on tumour progression. Both cancer cell lines showed reduced proliferation, migration and invasion, as epithelial-mesenchymal transition (EMT) was inactivated. In vivo therapeutic effect on the innate and adaptive immune processes was validated in a subcutaneous liver cancer model by the intratumoural injection of sevelamer. Tumour growth was significantly inhibited by the partial deprivation of intratumoural Pi as the tumour microenvironment under low-Pi stress is more immunostimulatory. The anticancer immune response, activated by low-Pi stress, suggests a new macrophage-based immunotherapeutic modality.

Low Pi stress enhances the sensitivity of hepatocellular carcinoma to sorafenib.

Sorafenib is a tyrosine kinase inhibitor for the treatment of advanced-stage HCC; however, clinical trials of sorafenib failed to demonstrate long-term survival benefits due to drug resistance. Low Pi stress has been shown to inhibit tumor growth and the expression of multidrug resistance-associated proteins. In this study, we investigated the sensitivity of HCC to sorafenib under conditions of low Pi stress. As a result, we found that low Pi stress facilitated sorafenib-mediated suppression of migration and invasion of HepG-2 and Hepa1-6 cells by decreasing the phosphorylation or expression of AKT, Erk and MMP-9. Angiogenesis was inhibited due to decreased expression of PDGFR under low Pi stress. Low Pi stress also decreased the viability of sorafenib-resistant cells by directly regulating the expression of AKT, HIF-1a and P62. In vivo drug sensitivity analysis in the four animal models showed a similar tendency that low Pi stress enhances sorafenib sensitivity in both the normal and drug-resistant models. Altogether, low Pi stress enhances the sensitivity of hepatocellular carcinoma to sorafenib and expands the indications for sevelamer.

Evaluation of the safety and efficacy of transarterial sevelamer embolization in a rabbit liver cancer model: A challenge on the size rule for vascular occlusion.

As the most efficient method to treat hepatocellular carcinoma in the immediate or advanced stage, transarterial chemoembolization (TACE) is coming into the era of microsphere (MP). Drug-eluting beads have shown their huge potential as an embolic agent and drug carrier for chemoembolization, but their sizes are strictly limited to be above 40 µm, which was considered to occlude vessels in a safe mode. microsphere smaller than 40 µm is easy to be washed out and transported to the normal liver lobe or other organs, causing severe adverse events and failed embolization. To determine whether sevelamer ultrafine particle (0.2-0.5 µm) is qualified as a safe and efficient embolic agent, we investigated the safety and therapeutic efficiency of transarterial sevelamer embolization (TASE) in the VX2 rabbit liver cancer model, aiming to challenge the "40 µm" rule on the selection criteria of the MP. In a four-arm study, blank bead (Callisphere, 100-300 µm), luminescent polystyrene microsphere (10, 100 µm), and sevelamer particle were transarterially administered to evaluate the threshold size of the MP size for intrahepatic or extrahepatic permeability. Another four-arm study was designed to clarify the safety and efficiency of preclinical transarterial sevelamer embolizationTASE tests over other techniques. Sham (saline), TASE, C-TACE, and D-TACE (n = 6) were compared in terms of serum chemistry, histopathology, and tumor necrosis ratio. In the first trials, the "40 µm" rule was detectable on the VX2 cancer model, but the regulation has no application to the new embolic agent as sevelamer ultrafine particles have not been found to leak out from the VX2 lesions, only found in the embolized vessels. Pathology proves that less viable tumor residue was found 2 weeks after the procedure, evidencing a better therapeutic outcome. No adverse events were found except for a short stress response. These results indicate that sevelamer is a safe and efficient embolic as an alternative to the current MP-based embolization therapy techniques.

Sevelamer arsenite nanoparticle as a Pi-responsive drug carrier and embolic agent for chemoembolization.

Arsenic trioxide (As2O3, ATO) has limited therapeutic benefit to treat solid tumors, whether used alone or in combination. Nanoscale drug delivery vehicles have great potential to overcome the limitation of the utility of ATO by rapid renal clearance and dose-limiting toxicity. Polymeric materials ranging from gelatin foam to synthetic polymers such as poly(vinyl alcohol) were developed for vascular embolic or chemoembolic applications. Recently, we have introduced sevelamer, an oral phosphate binder, as a new polymeric embolic for vascular interventional therapy. In this paper, sevelamer arsenite nanoparticle with a polygonal shape and a size of 50-300 nm, synthesized by anionic exchange from sevelamer chloride, was developed as a Pi-responsive bifunctional drug carrier and embolic agent for chemoembolization therapy. At the same arsenic dosage, sevelamer arsenite-induced severer tumor necrosis than ATO on the VX2 cancer model. In vitro tests evidenced that Pi deprivation by sevelamer could enhance ATO's anticancer effect. The results showed that ATO in Pi starvation reduced cell viability, induced more apoptosis, and diminished the mitochondrial membrane potential (Δψm) of cells since Pi starvation helps ATO to further down-regulate Bcl-2 expression, up-regulate Bax expression, enhance the activation of caspase-3 and increase the release of cytochrome c, and the production of excessive reactive oxygen species (ROS). Sevelamer arsenite not only plays a Pi-activated nano-drug delivery system but also integrated anticancer drug with embolic for interventional therapy. Therefore, our results presented a new administration route of ATO as well as an alternative chemoembolization therapy.

Pi-inducedin-situaggregation of sevelamer nanoparticles for vascular embolization.

Decades have witnessed rapid progress of polymeric materials for vascular embolic or chemoembolic applications. Commercially available polymeric embolics range from gelatin foam to synthetic polymers such as poly(vinyl alcohol). Current systems under investigation include tunable, bioresorbable microspheres composed of chitosan or poly(ethylene glycol) derivatives,in situgelling liquid embolics with improved safety profiles, and radiopaque embolics that are trackablein vivo. In this paper, we proposed a concept of 'responsive embolization'. Sevelamer, clinically proved as an inorganic phosphate binder, was ground into nanoparticles. Sevelamer nanoparticle is highly mobile and capable of swelling and aggregating in the presence of endogenous inorganic phosphate, thereby effectively occluding blood flow in the vessel as it was administered as an embolic agent for interventional therapy. Moreover, citrated sevelamer nanoparticles delayed the aggregation, preferable to penetrate deeply into the capillary system. On the rabbit VX2 liver cancer model, both sevelamer particles aggregates occlude the tumor feeding artery, but backflow was found for the pristine one, thereby citrate passivation of sevelamer nanoparticles endows it have potential from 'bench to bedside' as a new type of vascular embolic.

Intratumoral Pi deprivation benefits chemoembolization therapy via increased accumulation of intracellular doxorubicin.

It is a decade-long controversy that transarterial chemoembolization (TACE) has definite priority over transarterial embolization (TAE) in treating patients with hepatocellular carcinoma (HCC), since HCC cells are regularly resistant to chemotherapy by enhanced expression of proteins that confer drug resistance, and ABC transporters pump the intracellular drug out of the cell. We addressed this issue by modulating the chemo-environment. In an animal model, sevelamer, a polymeric phosphate binder, was introduced as an embolic agent to induce intratumoral inorganic phosphate (Pi) starvation, and trans-arterially co-delivered with doxorubicin (DOX). The new type of TACE was named as DOX-TASE. This Pi-starved environment enhanced DOX tumoral accumulation and retention, and DOX-TASE thereby induced more severe tumor necrosis than that induced by conventional TACE (C-TACE) and drug-eluting bead TACE (D-TACE) at the same dose. In vitro tests showed that Pi starvation increased the cellular accumulation of DOX in an irreversible manner and enhanced cytotoxicity and cell apoptosis by suppressing the expression of ABC transporters (P-glycoprotein (P-gp), BCRP, and MRP1) and the production of intracellular ATP. Our results are indicative of an alternative interventional therapy combining chemotherapy with embolization more effectively.

Grass carp (Ctenopharyngodon idellus) SHP2 suppresses IFN I expression via decreasing the phosphorylation of GSK3ß in a non-contact manner.

As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3ß at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2, MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3ß in a non-contact manner. Meanwhile CiGSK3ß interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3ß, and then it depressed the expression of IFN I via GSK3ß-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3ß and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3ß-TBK1 signal axis.

Grass carp (Ctenopharyngodon idellus) Cdc25a down-regulates IFN 1 expression by reducing TBK1 phosphorylation.

In vertebrates, TANK Binding Kinase 1 (TBK1) plays an important role in innate immunity, mainly because it can mediate production of interferon to resist the invasion of pathogens. In mammals, cell division cycle-25a (Cdc25a) is a member of the Cdc25 family of cell division cycle proteins. It is a phosphatase that plays an important role in cell cycle regulation by dephosphorylating its substrate proteins. Currently, many phosphatases are reported to play a role in innate immunity. This is because the phosphatases can shut down or reduce immune signaling pathways by down-regulating phosphorylation signals. However, there are no reports on fish Cdc25a in innate immunity. In this paper, we conducted a preliminary study on the involvement of grass carp Cdc25a in innate immunity. First, we cloned the full-length cDNA of grass carp Cdc25a (CiCdc25a), and found that it shares the highest genetic relationship with that of Anabarilius grahami through phylogenetic tree comparison. In grass carp tissues and CIK cells, the expression of CiCdc25a mRNA was up-regulated under poly (I:C) stimulation. Therefore, CiCdc25a can respond to poly (I:C). The subcellular localization results showed that CiCdc25a is distributed both in the cytoplasm and nucleus. We also found that CiCdc25a can down-regulate the expression of IFN 1 with or without poly (I:C) stimulation. In other words, the down-regulation of IFN1 by CiCdc25a is independent of poly (I:C) stimulation. Further functional studies have shown that the inhibition of IFN1 expression by CiCdc25a may be related to decrease of TBK1 activity. We also confirmed that the phosphorylation of TBK1 at Ser172 is essential for production of IFN 1. In short, CiCdc25a can interact with TBK1 and subsequently inhibits the phosphorylation of TBK1, thereby weakens TBK1 activity. These results indicated that grass carp Cdc25a down-regulates IFN 1 expression by reducing TBK1 phosphorylation.

The tyrosine kinase SRC of grass carp (Ctenopharyngodon idellus) up-regulates the expression of IFN I by activating TANK binding kinase 1.

In response to viral infections, various pattern recognition receptors (PRRs) are activated for the production of type I interferon (IFN I). As a center of these receptor responses, TANK binding kinase-1 (TBK1) activates interferon regulatory factor 3 (IRF3). SRC is a member of Src family kinases (SFK) which participates in TBK1-mediated IFN I signaling pathway. In mammals, the immunological function of SRC is depended on its interaction with TBK1. To date, SRC has not been studied in fish. In this paper, we cloned the ORF of grass carp (Ctenopharyngodon idellus) SRC (CiSRC). CiSRC has a closer relationship with Sinocyclocheilus rhinocerous SRC (SrSRC). The expression level of CiSRC was significantly up-regulated following poly (I:C) stimulation in grass carp tissues and cells. Subcellular localization results showed that CiSRC is located both in the cytoplasm and nucleus, while CiTBK1 is only located in the cytoplasm of CIK cells. When GFP-CiSRC and FLAG-CiTBK1 were co-transfected into CIK cells, we found that they were co-localized in the cytoplasm. GST-pulldown and Co-immunoprecipitation analysis revealed that CiSRC and CiSRC tyrosine kinase domain deletion mutant (SRC-ΔTyrkc) can interact with CiTBK1, respectively. CiSRC promotes the phosphorylation of CiTBK1. Furthermore, the phosphorylation of TBK1 is more strongly under poly (I:C) stimulation. We also demonstrated that SRC can up-regulate IFN I expression. These results above unraveled that CiSRC initiates innate immune response by binding to and then up-regulating the phosphorylation of TBK1.

Grass carp (Ctenopharyngodon idella) TNK1 modulates JAK-STAT signaling through phosphorylating STAT1.

TNK1 (thirty-eight-negative kinase 1) belongs to the ACK (Activated Cdc42 Kinases) family of intracellular non-receptor tyrosine kinases that usually acts as an important regulator in cytokine receptor-mediated intracellular signal transduction pathways. JAK-STAT signal pathway acts as a key point in cellular proliferation, differentiation and immunomodulatory. Mammalian TNK1 is involved in antiviral immunity and activation of growth factors. However, TNK1 has rarely been studied in fish. To evaluate the role of fish TNK1 in JAK-STAT pathway, we cloned the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) TNK1 (CiTNK1). CiTNK1 protein consists of N-terminal Tyrkc (tyrosine kinase) domain, C-terminal SH3 (Src homology 3) domain and Pro-rich domain. Phylogenetic analysis showed that CiTNK1 has a closer relationship with Danio rerio TNK1. The expression and phosphorylation of CiTNK1 in grass carp tissues and cells was increased under poly(I:C) stimulation. Subcellular localization and co-immunoprecipitation indicated that CiTNK1 is targeted in the cytoplasm and interacts with grass carp STAT1 (CiSTAT1). Co-transfection of CiTNK1 and CiSTAT1 into cells facilitated the expression of IFN I. This is because that the presence of CiTNK1 enhanced the phosphorylation of CiSTAT1 and causes activation of CiSTAT1. Our results revealed that TNK1 can potentiate the phosphorylation of STAT1 and then regulates JAK-STAT pathway to trigger IFN I expression in fish.

Grass carp Mre11A activates IFN 1 response by targeting STING to defend against GCRV infection.

Mre11A is considered as a cytosolic DNA receptor in mammals. However, it is rarely known about Mre11A in other vertebrates. Recently, a mammalian ortholog of Mre11A has been identified in grass carp (Ctenopharyngodon idellus) in our lab. Phylogenetic-tree analysis provided evidence for a close genetic relationship between C.idellus Mre11A and Carassius auratus Mre11A. The tissue expression profile of CiMre11A was detected, with a relatively higher level of expression in kidney, intestines, liver and spleen than that in other tissues after grass carp reovirus (GCRV) infection. Similarly, CiMre11A was also up-regulated in CIK cells after treatment with GCRV. Q-PCR and dual-luciferase assays indicated that the transcription levels of IFN1 and ISG15 were inhibited by CiMre11A knockdown, but were gradually augmented after CIK cells were transfected with increasing amounts of CiMre11A. Subcellular localization assays showed that a part of CiMre11A was translocated from the nucleus to the cytoplasm. Co-immunoprecipitation and co-localization assays demonstrated that CiMre11A interacts with CiSTING in response to GCRV infection. In CIK cells, the expressions of both IFN1 and ISG15 were acutely up-regulated by CiMre11A overexpression, as well as by co-overexpression of CiMre11A and CiSTING. CiMre11A and CiSTING induced the phosphorylation and cytoplasmic-to-nuclear translocation of IRF7 in CIK cells. The multiplication of GCRV in CIK cells was inhibited by the overexpression of CiMre11A and CiSTING.

Grass carp (Ctenopharyngodon idella) GPATCH3 initiates IFN 1 expression via the activation of STING-IRF7 signal axis.

GPATCH3, a protein with G-patch domain, is known to participate in innate immune response and organ development in mammals. However, there are few reports on GPATCH3 in fish. Here the cDNA sequence of GPATCH3 was cloned from Ctenopharyngodon idella (CiGPATCH3, MN149902) and was determined its character. A cDNA sequence of CiGPATCH3 is 1646 bp and contains an ORF of 1221 bp translating a protein of 407 amino acids. Phylogenetic analysis uncovered that CiGPATCH3 possesses a relatively high degree of homology with Cyprinus carpio GPATCH3. The mRNA level of CiGPATCH3 was increased following the intracellular stimulation of poly (I:C) into CIK cells. In vivo, over-expression of CiGPATCH3 can significantly up-regulate IFN 1 and ISG15 expression at mRNA and protein levels. To investigate the molecular mechanism by which GPATCH3 initiates the innate immune response in fish, co-IP experiments were performed to analyze the substrates of CiGPATCH3. The results showed that CiGPATCH3 directly interacted with CiSTING, but not with CiIRF3, CiIRF7, CiTBK1 or CiIPS-1. As compared with the single transfection of CO cells with either CiGPATCH3 or CiSTING, the expression of IFN 1 was more significantly up-regulated in cells under treatment with dual transfection of CiGPATCH3 and CiSTING. Knockdown of CiGPATCH3 inhibited STING-mediated IFN 1 expression in fish cells. Over-expression of CiGPATCH3 and CiSTING facilitated the phosphorylation and cytoplasmic-to-nuclear translocation of CiIRF7. These results explicitly showed that CiGPATCH3 up-regulates IFN 1 and ISG15 expression via the activation of STING-IRF7 signal axis in vivo.

IRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis in fish.

As a NAD+-dependent deacetylase, SIRT1 is widely involved in apoptosis and cellular inflammation via multiple pathways such as p53, NF-кB and STAT. More and more studies have shown that p53 is the first non-histone deacetylation target of SIRT1. SIRT1-p53 axis thus plays an important role in mammalian cells. IRF9 is an important member of interferon regulator factor family and performs an important role in innate immunity against foreign virus invasion. More importantly, human IRF9 can suppress the SIRT1-p53 axis. However, the functions and relationship between IRF9 and SIRT1-p53 axis are rarely studied in fish. To this end, we made a preliminary research on the functions of grass carp (Ctenopharyngodon idella) IRF9, SIRT1 and p53 in apoptosis and innate immunity. Firstly, we cloned and identified the ORF of SIRT1 (named CiSIRT1, MN125614) from C. idella and demonstrated that CiIRF9 promoted apoptosis, while CiSIRT1 inhibited apoptosis by flow cytometry and TUNEL experiments. Next, we found the interaction between CiSIRT1 and Cip53 in vivo by co-immunoprecipitation experiments. Moreover, the colocalization analysis also showed CiSIRT1 and Cip53 were mainly distributed in nucleus. Thirdly, we got a conclusion that CiIRF9 can repress the expression of CiSIRT1, implying that CiIRF9 regulates CiSIRT1-p53 axis. Finally, CiSIRT1 mRNA level was significantly up-regulated and the expression reached the highest level at 24 h post poly (I:C) stimulation in CIK cells. So, CiSIRT1 may exert an important function in innate immunity. Furthermore, we found CiSIRT1 down-regulated the expression of CiIFN1. In summary, CiIRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis. These findings will provide a new theoretical basis for the research on teleost innate immunity.

Grass carp (Ctenopharyngodon idella) IRAK1 and STAT3 up-regulate synergistically the transcription of IL-10.

In vertebrates, IL-10 is an anti-inflammatory factor that serves as a key inhibitory role in a wide range of immune responses. IRAK1 (IL-1 receptor-associated kinase 1), a key molecule in the inflammatory signal of IL-1R/TLR, plays an important pivotal role in regulating the autoimmunity of body. STAT3 (Signal transducer and activator of transcription 3) activated by IRAK1 participates in inflammation, tumorigenesis, metabolic disorders and immune response. Under the stimulation of LPS, IRAK1 enters the nucleus to form a dimer with STAT3 and regulates the expression of IL-10. However, the relationship between fish IRAK1 and STAT3 has not been reported. To explain the anti-inflammation in fish, we amplified and identified the complete open reading frame of grass carp IRAK1 (CiIRAK1) and STAT3 (CiSTAT3) based on the existing sequences. The expression of CiIRAK1 and CiSTAT3 were up-regulated significantly under the stimulation of LPS. This result suggests that both CiIRAK1 and CiSTAT3 may be involved in LPS-induced TLR4 pathway. The subcellular localization experiment revealed that CiIRAK1 is distributed in cytoplasm and enters nucleus after LPS stimulation. CiSTAT3 is distributed in both cytoplasm and nucleus with or without LPS stimulation. Immunoprecipitation assay revealed that CiIRAK1 interacted with CiSTAT3 under LPS stimulation. However in absence of LPS stimulation, CiIRAK1 and CiSTAT3 cannot interact with each other. Subsequently, immunofluorescence colocalization experiment further proved the interaction of CiIRAK1 and CiSTAT3 in nucleus under LPS stimulation. The dual luciferase reporter assays indicated that the binding of CiIRAK1 and CiSTAT3 synergistically enhanced the activity of CiIL-10 promoter.