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Non-alcoholic fatty liver disease (NAFLD) is the commonest reason for chronic liver diseases in the world and is commonly related to the hepatic manifestation of the metabolic syndrome. Non-alcoholic steatohepatitis (NASH) is a deteriorating form of NAFLD, which can eventually develop into fibrosis, cirrhosis, and liver cancer. The reason for NAFLD/NASH development is complicated, such as liver lipid metabolism, oxidative stress, inflammatory response, apoptosis and autophagy, liver fibrosis and gut microbiota. Apart from bariatric surgery and lifestyle changes, officially approved drug therapy for NAFLD/NASH treatment is lacking. Salidroside (SDS) is a phenolic compound extensively distributed in the tubers of Rhodiola plants, which possesses many significant biological activities. This review summarized the related targets regulated by SDS in treating NAFLD/NASH. It is indicated that SDS could improve the status of NAFLD/NASH by ameliorating abnormal lipid metabolism, inhibiting oxidative stress, regulating apoptosis and autophagy, reducing inflammatory response, alleviating fibrosis and regulating gut microbiota. In conclusion, although the multiple bioactivities of SDS have been confirmed, the clinical data are inadequate and need to become the focus of attention in the later study.

Conventional role of ribosomal proteins is ribosome assembly and protein translation, but some ribosomal proteins also show antimicrobial peptide (AMP) activity, though their mode of action remains ill-defined. Here we demonstrated for the first time that amphioxus RPS15, BjRPS15, was a previously uncharacterized AMP, which was not only capable of identifying Gram-negative and -positive bacteria via interaction with LPS and LTA but also capable of killing the bacteria. We also showed that both the sequence and 3D structure of RPS15 and its prokaryotic homologs were highly conserved, suggesting its antibacterial activity is universal across widely separated taxa. Actually this was supported by the facts that the residues positioned at 45-67 formed the core region for the antimicrobial activity of BjRPS15, and its prokaryotic counterparts, including Nitrospirae RPS1933-55, Aquificae RPS1933-55 and P. syringae RPS1950-72, similarly displayed antibacterial activities. BjRPS15 functioned by both interaction with bacterial surface via LPS and LTA and membrane depolarization as well as induction of intracellular ROS. Moreover, we showed that RPS15 existed extracellularly in amphioxus, shrimp, zebrafish and mice, hinting it may play a critical role in systematic immunity in different animals. In addition, we found that neither BjRPS15 nor its truncated form BjRPS1545-67 were toxic to mammalian cells, making them promising lead molecules for the design of novel AMPs against bacteria. Collectively, these indicate that RPS15 is a new member of AMP with ancient origin and high conservation throughout evolution.

Inflammatory response is an innate host defense mechanism, and its regulation is essential for the host to get rid of harm by the excessive reactions. We first utilized proteomics approach to identify amphioxus humoral fluid proteins in response to LPS-induced inflammation. A total of 26 differentially expressed proteins, mainly involved in energy metabolism and cytoskeleton rearrangement processes, were identified between LPS-treated and control animals. Furthermore, we found a single uncharacterized protein (termed BjIM1) out of the most up-regulated ones, and examined its role in the regulation of immune and inflammatory responses. BjIM1 is predominantly expressed in the hepatic caecum, and its promoter sequence includes many binding sites for immune-relevant transcription factors. Importantly, recombinant BjIM1 (rBjIM1) is able to inhibit LPS-induced up-regulation of TLR pathway genes, such as MyD88, IKK, NF-κB1, Rel, p38, JNK and AP-1, indicating that BjIM1 may negatively regulate the TLR signaling pathway in amphioxus. Moreover, rBjIM1 also modulates the expression of genes involved in the interaction network of inflammation, energy metabolism and cytoskeleton rearrangement, including SIRT1, Rac1 and NOX2, in the LPS-induced inflammatory response in amphioxus. Collectively, our studies suggest that BjIM1 is an uncharacterized protein functioning as a modulator of inflammatory networks in amphioxus.

Previous studies have shown that heat shock proteins (Hsps) are broadly associated in immune responses in a variety of animals. However, it remains largely unknown about the direct roles of Hsps during a bacterial infection. In this study, we have cloned and characterized the cDNAs of two Hsp genes in the amphioxus Branchiostoma japonicum, termed Bjhsp5 and Bjhsp90α, the first ones in this evolutionarily important animal. Both Bjhsp5 and Bjhsp90α showed distinct tissue expression patterns, and were inducible by challenge with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), suggesting they may be involved in anti-infectious responses. We also showed that both BjHsp5 and BjHsp90α displayed lectin-like property with affinity to both the Gram-negative and -positive bacteria as well as their signature molecules LPS and LTA, hinting they may both act as a pattern recognition receptor, capable of identifying pathogens. In addition, we found that BjHsp5 and BjHsp90α were both able to agglutinate the Gram-negative and -positive bacteria in the presence of Ca2+, suggesting they may be able to trap the invading pathogens together in vivo, avoiding them moving around and thereby protecting the host from pathogenic attack. These data provide a new angle to the roles of Hsps in immune defense.

The vertebrate liver is regarded as an organ essential to the regulation of immunity and inflammation as well as being central to the metabolism of nutrients. Here, we discuss the functions that the hepatic cecum of amphioxus plays in the regulation of immunity and inflammation, and the molecular basis of this. It is apparent that the hepatic cecum performs important roles in the immunity of amphioxus including immune surveillance, clearance of pathogens and acute phase response. Therefore, the hepatic cecum, like the vertebrate liver, is an organ functioning as a key integrator of immunity in amphioxus.

Previous studies show that some ribosomal proteins possess antimicrobial peptide (AMP) activity. However, information as such remains rather fragmentary and limited. Here we demonstrated for the first time that amphioxus RPS15, BjRPS15, was a previously uncharacterized AMP, which was not only capable of identifying Gram-negative and -positive bacteria via interaction with LPS and LTA but also capable of killing the bacteria. We also showed that both the sequence and 3D structure of RPS15 and its prokaryotic homologs were highly conserved, suggesting its antibacterial activity is universal across widely separated taxa. Actually this was supported by the facts that the residues positioned at 45-67 formed the core region for the antimicrobial activity of BjRPS15, and its prokaryotic counterparts, including Nitrospirae RPS1933-55, Aquificae RPS1933-55 and P. syringae RPS1950-72, similarly displayed antibacterial activities. BjRPS15 functioned by both interaction with bacterial membrane via LPS and LTA and membrane depolarization as well as induction of intracellular ROS. Moreover, we showed that RPS15 existed extracellularly in amphioxus, shrimp, zebrafish and mice, hinting it may play a critical role in systematic immunity in different animals. In addition, we found that neither BjRPS15 nor its truncated form BjRPS1545-67 were toxic to mammalian cells, making them promising lead molecules for the design of novel peptide antibiotics against bacteria. Collectively, these indicate that RPS15 is a new member of AMP with ancient origin and high conservation throughout evolution.Author summaryRibosomal protein, a component of ribonucleoprotein particles, is traditionally known involved in protein synthesis in a cell. Here we demonstrated for the first time that amphioxus ribosomal protein 15 was a novel antibacterial protein, capable of recognizing Gram-negative and -positive bacteria as well as killing them. It killed the bacteria by a combined mode of action of disrupting bacterial membrane integrity and inducing radical oxygen species production. We also showed that both eukaryotic ribosomal protein 15 and its prokaryotic counterpart ribosomal protein 19 possessed antibacterial activity, indicating that the antibacterial property is universal for this family of molecules. Moreover, we found that ribosomal protein 15 was present in the circulation system of various animals including shrimp, amphioxus, zebrafish and mice, suggesting it may physiologically play a key role in systematic immunity. Altogether, our study provides a new angle for understanding the biological function of ribosomal proteins.

Cofilin-1 (Cfl1), a member of the ADF/cofilin family, has been identified as one of differentially expressed proteins in human dendritic cells challenged with lipopolysaccharide (LPS), suggesting that it may be involved in immune response. Here we showed that zebrafish cfl1 was markedly up-regulated by LPS and LTA treatment. We also showed that zebrafish recombinant Cfl1 (rCfl1) not only bound to the Gram-negative and positive bacteria A. hydrophila and S. aureus as well as their signature molecules LPS and LTA but also inhibited the growth of the bacteria. Moreover, we found that the heparin-binding motif-containing regions of Cfl1, i.e., Cfl19-25, Cfl134-51 and Cfl1108-125, like rCfl1, were also able to bind to LPS and LTA and to inhibit the bacterial growth. rCfl1, Cfl19-25, Cfl134-51, and Cfl1108-125 were all able to cause bacterial cell destruction, to induce membrane depolarization, and to stimulate intracellular ROS production. Finally, we showed that zebrafish Cfl1 could protect developing embryos/larvae against attack by the potential pathogen A. hydrophila. These data together indicate that zebrafish Cfl1 plays an immune-relevant role as a newly-characterized antimicrobial protein.

Previous studies have shown that protein disulfide isomerase (PDI), a member of the thioredoxin (TRX) superfamily, are broadly associated with immune responses in a variety of animals. However, it remains largely unknown about the direct roles of PDIs during a bacterial infection. In this study, we identified the presence of a single pdi gene in the amphioxus Branchiostoma japonicum, Bjpdi. The deduced protein BjPDI is structurally characterized by the presence of four Trx-like domains in the order of a, b, b’ and a’ and a short acidic C-terminal tail, that are characteristic of PDIs. We demonstrated that rBjPDI displayed both thiol reductase and disulfide bond isomerase activities, indicating comparability of BjPDI with PDIs in term of enzymatic activities. We also showed that rBjPDI induced bacterial agglutination and exhibited a lectin-like activity capable of binding both bacteria (E. coli and S. aureus) and their signature molecules LPS and LTA. Furthermore, BjPDI could kill S. aureus via inducing membrane depolarization and intracellular ROS production in vitro, and treatment of amphioxus with a blocking anti-PDI antibody in vivo markedly reduced the survival rate of amphioxus following attack by S. aureus. Collectively, our study demonstrates that amphioxus protein disulfide isomerase acts as both an enzyme and an immunocompotent factor, and reports the specific function and mode of action of PDIs in immune responses.

Low-density lipoprotein receptor-related protein (LRP) is a group of important endocytic receptors contributing to binding ligands and maintaining internal environment. In this study, we identified a soluble LRP-like molecule in the amphioxus B. japonicum, BjLRP, with an uncharacterized domain structure combination of LY-EGF-CRD-EGF-CRD. It was mainly expressed in the gill, muscle, notochord and testis, and was significantly up-regulated following the challenge with bacteria. Recombinant BjLRP was capable of interacting with both Gram-negative and positive bacteria as well as PAMPs including lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Interestingly, recombinant LY peptide was also able to bind to the Gram-negative and positive bacteria as well as the PAMPs LPS, LTA and PGN. By contrast, none of recombinant EGF1, EGF2, CRD1 and CRD2 had affinity to the bacteria and the PAMPs. In addition, BjLRPΔLY had no affinity to the PAMPs, although BjLRPΔLY showed slight affinity to the bacteria. These suggest that the interaction of BjLRP with the bacteria and PAMPs was primarily attributable to the LY domain. It is clear that BjLRP is a novel pattern recognition protein capable of identifying and interacting with invading bacteria in amphioxus.

A complement system operating via the alternative pathway similar to that of vertebrates has been demonstrated in the primitive chordate amphioxus. However, the factor P (fP), a positive regulator of the alternative pathway, remains elusive in amphioxus to date. In this study, we identified and characterized a properdin gene in the amphioxus B. japonicum, BjfP, which represents an archetype of vertebrate properdins. Real-time PCR analysis showed that the BjfP was ubiquitously expressed and its expression was significantly up-regulated following the challenge with bacteria or lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Recombinant BjfP (rBjfP) and its truncated proteins including rTSR1-3, rTSR4-6 and rTSR7-8, were all capable of interacting with both Gram-negative and positive bacteria as well as LPS and LTA. Moreover, rBjfP, rTSR1-3 and rTSR4-6 could also specifically bind to C3b. Importantly, both rTSR1-3 and rTSR4-6 could inhibit the binding of rBjfP to C3b, and thus suppress the activation of the alternative pathway of complement, suggesting the involvement of BjfP in the alternative pathway. This is the first report showing that a properdin protein in invertebrates plays similar roles to vertebrate properdins. Collectively, these data suggest that BjfP might represent the ancient molecule from which vertebrate properdins evolved.

Klotho, a putative aging suppressor, shares sequence similarity with members of the glycosidase family 1. It has been identified in several vertebrate species, but only mouse Klotho has so far been proven to exhibit β-glucuronidase activity. Thus, the argument that Klotho from animals other than mouse has glycosidase activity remains open. Moreover, little information is available regarding the structure-activity relationship of Klotho. Here, we demonstrate the presence of a single klotho gene in the amphioxus Branchiostoma japonicum, Bjklotho, which possesses two tandem domains named BjKL1 and BjKL2, and each of them has two glutamic acid residues that have been shown to be involved in the catalytic activity of family 1 glycosidase. Enzymatic activity assays of the recombinant proteins BjKL1 and BjKL2 revealed that only BjKL2 displayed β-glucosidase activity, but BjKL1 did not. Structural analysis showed that there existed nine consecutive but not conserved residues in the β6α6 loop, which affects the conformational form in the entrance to the catalytic pocket of BjKL1 and BjKL2, thereby leading to a subtle difference in the enzyme-substrate binding and interaction. Furthermore, the substitution of the nine residues 354QNRVDPNDT362 in BjKL1 by the residues 884EDNVVVGAA892 in BjKL2 resulted in significant increase in β-glucosidase activity in the BjKL1 mutant. Our results indicate that BjKL2 possesses β-glucosidase, the first data as such in invertebrates. We also identify, for the first time, the residues 884EDNVVVGAA892 in BjKL2 a sequence critical and indispensable for glucosidase.

Midkine (MK) and pleiotrophin (PTN) are the only two members of heparin-binding growth factor family. MK/PTN homologues found from Drosophila to humans are shown to have antibacterial activities and their antibacterial domains are conserved during evolution. However, little is known about MK/PTN homologue in the basal chordate amphioxus, and overall, information regarding MK/PTN homologues is rather limited in invertebrates. In this study, we identified a single MK/PTN homologue in Branchiostoma japonicum, termed BjMiple, which has a novel domain structure of PTN-PTNr1-PTNr2, and represents the ancestral form of vertebrate MK/PTN family proteins. BjMiple was expressed mainly in the ovary in a tissue-dependent fashion, and its expression was remarkably up-regulated following challenge with bacteria or their signature molecules LPS and LTA, suggesting its involvement in antibacterial responses. Functional assays revealed that BjMiple had strong antimicrobial activity, capable of killing a panel of Gram-negative and Gram-positive bacteria via a membranolytic mechanism, including interaction with bacterial membrane via LPS and LTA, membrane depolarization and high intracellular levels of ROS. Importantly, strong antibacterial activity was localized in PTN42-61 and PTNr142-66. Additionally, BjMiple and its derived peptides PTN42-61 and PTNr142-66 were not cytotoxic to human RBCs and mammalian cells. Taken together, our study suggests that amphioxus Miple is the ancestral type of vertebrate MK/PTN family homologues, and can play important roles as innate peptide antibiotics, which renders it a promising template for the design of novel peptide antibiotics against multi-drug resistant bacteria.

Fish vitellogenin (Vg) has been shown to mediate the phagocytosis via interaction with a Fcγ-like phagocytic receptor on macrophages, but identification of such a receptor and its functional characterization remains lacking. In this study, we isolated a cDNA of polymeric immunoglobulin receptor (pIgR) from sea bass, which encoded a single-spanning transmembrane protein of 326 amino acids including a 21-amino acid signal peptide, an extracellular region, a transmembrane region and a 36-amino acid intracellular region included two Ig-like domains (ILDs), and was expressed in multiple lymphoid organs. We then showed that recombinant extracellular domain of sea bass pIgR was capable of binding to Vg as well as IgG and IgM. We also showed that Vg as well as IgG and IgM interacted with pIgR-expressing HEK 293T cells. Importantly, we demonstrated that Vg as well as IgG and IgM were all capable of enhancing phagocytosis by HEK 293T cells and inducing expression of tnf-α and il-1β, via interacting with pIgR. Collectively, these results suggest that fish Vg, analogous to IgG and IgM, can interact with pIgR and result in similar down-stream immune responses, providing an additional evidence that Vg plays an antibody-like role.

Over 1200 C-type lectin gene models have been identified in amphioxus, but only a few of them have been functionally characterized. In this study, we identified a C-type lectin, BjCTL, with domain structure of LDLa-CTLD-EGF_Lam, the first such data in chordates. It was expressed mainly in the notochord and ovary in a tissue-dependent fashion. Recombinant BjCTL was characterized as a typical Ca2+-dependent carbohydrate-binding protein capable of agglutinating and binding to both Gram-negative and positive bacteria we tested. In addition, it specifically bound to insoluble lipopolysaccharide, lipoteichoic acid and peptidoglycan, which can be inhibited by galactose. We also showed that the interaction of BjCTL with the bacteria is primarily attributable to CTLD domain. Thus, BjCTL is a novel pattern recognition protein involved in lectin-mediated innate immunity.