Potential immune-related therapeutic mechanisms of multiple traditional Chinese medicines on type 2 diabetic nephropathy based on bioinformatics, network pharmacology and molecular docking.

BACKGROUND: The prevalence of type 2 diabetic nephropathy (T2DN) ranges from 20 % to 40 % among individuals with type 2 diabetes. Multiple immune pathways play a pivotal role in the pathogenesis of T2DN. This study aimed to investigate the immunomodulatory effects of active ingredients derived from 14 traditional Chinese medicines (TCMs) on T2DN. METHODS: By removing batch effect on the GSE30528 and GSE96804 datasets, we employed a combination of weighted gene co-expression network analysis, least absolute shrinkage and selection operator analysis, protein-protein interaction network analysis, and the CIBERSORT algorithm to identify the active ingredients of TCMs as well as potential hub biomarkers associated with immune cells. Functional analysis was conducted using Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and gene set variation analysis (GSVA). Additionally, molecular docking was employed to evaluate interactions between active ingredients and potential immunotherapy targets. RESULTS: A total of 638 differentially expressed genes (DEGs) were identified in this study, comprising 5 hub genes along with 4 potential biomarkers. Notably, CXCR1, CXCR2, and FOS exhibit significant associations with immune cells while displaying robust or favorable affinities towards the active ingredients kaempferol, quercetin, and luteolin. Furthermore, functional analysis unveiled intricate involvement of DEGs, hub genes and potential biomarkers in pathways closely linked to immunity and diabetes. CONCLUSION: The potential hub biomarkers and immunotherapy targets associated with immune cells of T2DN comprise CXCR1, CXCR2, and FOS. Furthermore, kaempferol, quercetin, and luteolin demonstrate potential immunomodulatory effects in modulating T2DN through the regulation of CXCR1, CXCR2, and FOS expression.

Low-complexity domain-containing protein (LCDP), induced accumulation of calcium carbonate individual crystals to irregular polycrystals in the triangle sail mussel Hyriopsis cumingii.

The nacre layer is composed of sheet-like aragonite crystals, with often loosely arranged polycrystal aragonite sheets which may induce poor mechanical properties in shells. In this study, a full-length low-complexity domain-containing protein (LCDP) cDNA from the triangle sail mussel Hyriopsis cumingii was generated and its role in shell formation investigated. The full-length cDNA was 1058 bp; it had an open reading frame (ORF) of 714 bp encoding 237 amino acids and contained a 20-amino acid signal peptide at the N-terminus and two low-complexity domains. H. cumingii LCDP was not homologous with other species. Tissue expression analyses showed that LCDP was specifically expressed in the mantle. In shell repair assays, significantly higher LCDP expression was observed in the shell repair group from days 12-21 (p < 0.01). After LCDP silencing, aragonite flake shapes in pearl layers became irregular with disordered deposition, while calcium carbonate (CaCO3) crystal surfaces in prismatic layers became rougher and organic matrices between crystals appeared skeletonized, indicating the importance of biomineralization. Our in vitro CaCO3 crystallization assays showed that LCDP induced single crystals to polycrystals, probably via loose arrangement between aragonite flakes. These results provide new insights on freshwater mollusk biomineralization and a theoretical basis for improved pearl quality.

Glutamine exerts a protective effect on osteoarthritis development by inhibiting the Jun N-terminal kinase and nuclear factor kappa-B signaling pathways.

Strategies for treating osteoarthritis (OA) have become a research focus because an effective treatment for OA is unavailable. The objective of this study was to explore the effects and underlying mechanisms of glutamine (Gln) in OA. First, the chondrocytes were identified and a standard IL-1ß-induced OA model was established. After treatment with Gln or saline, the viability and apoptosis of chondrocytes were evaluated using a CCK-8 assay and flow cytometry analysis, which revealed that Gln can improve the IL-1ß-induced OA cells. Meanwhile, Gln can enhance the expression of aggrecan and collagen II, which are protective proteins for articular cartilage. Instead, Gln inhibited the expression of matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-13 (MMP-13), which can degrade cartilage. To better understand the underlying mechanisms of Gln in IL-1ß-induced chondrocytes, the classical OA pathways of JNK and NF-κB were examined at the protein and mRNA levels using western blot and qRT-PCR analyses. We found that JNK and NF-κB were downregulated gradually depending on the Gln dose and protective and destructive factors changed based on changes of JNK and NF-κB. The effects of high-dose Gln were more effective than low-dose. Moreover, Gln was applied to the animal OA model to check the effects in vivo. The results showed that Gln attenuated cartilage degeneration and decreased OARSI scores, which demonstrated that Gln can improve OA. The experiments showed that Gln can benefit mice with OA by inhibiting the JNK and NF-κB signaling pathways.

Enhanced adsorption capacity of tetracycline on tea waste biochar with KHCO3 activation from aqueous solution.

Activation is an important pathway that can enhance the adsorption capacity of biochar. In this study, a modified tea waste biochar (MTWBC) was prepared via a two-step pyrolysis approach with KHCO3 activation. Pristine tea waste biochar (TWBC) was also produced as control via one-step pyrolysis without activation. Various characterizations were undertaken to investigate the influence of modification on the morphology, composition, carbon structure, surface area, and functional group of biochar, including scanning electron microscope (SEM), surface area and pore analyzer, element analysis, point of zero charge (pHPZC), X-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). After KHCO3 activation treatment, the surface area, total pore volume, and micropore volume of MTWBC reached 1981 m2·g-1, 0.8547 cm3·g-1, and 0.6439 cm3·g-1 which were 7.34-fold, 7.27-fold, and 7.30-fold increases, respectively, compared with TWBC. The aromaticity, hydrophilicity, and polarity of the MTWBC increased after modification. More graphitization with less defective structures occurred in MTWBC after modification. The C-, O-, and N-containing groups in MTWBC also changed after the reaction of KHCO3. The pseudo-second-order and Freundlich models best described the adsorption process on biochar. The maximum adsorption capacity of tetracycline (TC) on MTWBC reached 293.46 mg·g-1, which was 15-fold more than that of TWBC (19.68 mg·g-1). An alkaline environment decreased the TC adsorption on biochars. The presence of Na+, K+, Ca2+, and Mg2+ inhibited TC adsorption onto biochars. The influence of Cu2+ on TC adsorption by biochars depends on its initial concentration. The enhanced adsorption capacity of TC on MTWBC was mainly attributable to the large surface area, the improved pore volume, and more aromatic structure. The adsorption mechanism was based on pore filling and π-π EDA interaction. Therefore, KHCO3 activated biochar has the potential to remove TC from aquatic environments.

RNAi-mediated knock-down of the dopamine beta-hydroxylase gene changes growth of razor clams.

Dopamine beta-hydroxylase (DßH) plays an essential role in the synthesis of catecholamines (CA) in neuroendocrine networks. In the razor clam, Sinonovacula constricta a novel gene for DßH (ScDßH-α) was identified that belongs to the copper type II ascorbate-dependent monooxygenase family. Expression analysis revealed ScDßH-α gene transcripts were abundant in the liver and expressed throughout development. Knock-down of ScDßH-α in adult clams using siRNA caused a reduction in the growth rate compared to control clams. Reduced growth was associated with strong down-regulation of gene transcripts for the growth-related factors, platelet derived growth factors A (PDGF-A) (P < 0.001) 24 h after ScDßH-α knock-down, vascular endothelial growth factor (VEGF1) (P < 0.001) and platelet derived growth factor B (PDGF-B-2) (P < 0.001) 24 h and 48 h after ScDßH-α knock-down and transforming growth factor beta (TGF-ß1) (P < 0.001) 48 h and 72 h after ScDßH-α knock-down. Taken together the results suggest that the novel ScDßH-α gene through its role in CA synthesis is involved in growth regulation in the razor clam and possibly other bivalves.

Dopamine beta-hydroxylase and its role in regulating the growth and larval metamorphosis in Sinonovacula constricta.

Dopamine beta-hydroxylase (DßH) plays a key role in the synthesis of catecholamines (CAs) in the neuroendocrine regulatory network. The DßH gene was identified from the razor clam Sinonovacula constricta and referred to as ScDßH. The ScDßH gene is a copper type II ascorbate-dependent monooxygenase with a DOMON domain and two Cu2_monooxygen domains. ScDßH transcript expression was abundant in liver and hemolymph. During early development, ScDßH expression significantly increased at the umbo larval stage. Furthermore, the inhibitors and siRNA of DßH were screened. After challenge with DßH inhibitor, the larval metamorphosis and survival rates, and juvenile growth were obviously decreased. Under the siRNA stress, the larval metamorphosis and survival rates were also significantly decreased. Therefore, ScDßH may play an important regulating role in larval metamorphosis and juvenile growth.

A novel nacre matrix protein hic24 in Hyriopsis cumingii is essential for calcium carbonate nucleation and involved in pearl formation.

Matrix proteins play important roles in molluscan shell biomineralization, which helps in the understanding of mechanisms associated with pearl formation. In this study, we characterized the gene encoding a novel shell-matrix protein, hic24, in Hyriopsis cumingii and investigated its structure and function. The full cDNA sequence of hic24 is 756 bp, with an open reading frame of 654 bp encoding 217 amino acids, including a signal peptide of 18 amino acids. Sequence analysis revealed that the protein is ∼23.5 kDa, and that Gly accounted for 11.5% of the total amino acid content. Secondary structure prediction indicated a structure comprised predominantly by ß-folds. Quantitative real-time polymerase chain reaction and in situ hybridization indicated that hic24 is expressed in the dorsal epithelial cells of the mantle, indicating hic24 as a nacreous-layer matrix protein. Additionally, hic24 expression patterns during pearl biomineralization showed that hic24 regulates the growth of the later nacreous layer. After attenuating hic24 expression by RNA interference in the mantle, we observed that hic24 plays a role in biomineralization of the shell nacre by inhibiting calcium carbonate nucleation.

Identification of tumor necrosis factor receptor-associated factor 6 in the pearl mussel Hyriopsis cumingii and its involvement in innate immunity and pearl sac formation.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) acts as a central intracellular signal adapter molecule that mediates the tumor necrosis factor receptor superfamily and the interleukin-1 receptor/Toll-like receptor family in vertebrates and invertebrates. In the present study, HcTRAF6, a molluscan homologue of TRAF6 from Hyriopsis cumingii, has been cloned and identified. The entire open reading frame of HcTRAF6 was found to comprise a 1965-bp region that encodes a predicted protein of 654 amino acids, which contains conserved characteristic domains including a RING domain, two TRAF-type zinc finger domains, a typical coiled coil and the MATH domain. Phylogenetic analysis revealed that HcTRAF6 was aggregated closely with CsTRAF6 from Cyclina sinensis in the invertebrate cluster of mollusks. Further, qRT-PCR analysis showed that HcTRAF6 mRNA was extensively distributed in mussel tissues with a high expression in gills. After immune stimulation with Aeromonas hydrophila and lipopolysaccharides, the transcription of HcTRAF6 was obviously induced in the gills and hemocytes. In addition, significant fluctuation in HcTRAF6 expression was observed in the pearl sac, gills and hemocytes after mantle implantation. These findings confirmed its role in the alloimmune response. Dual-luciferase reporter assay showed that over-expression of HcTRAF6 could enhance the activity of the NF-κB reporter in a dose-dependent manner. Further, the RNA interference showed that the up-regulation of antimicrobial peptides in anti-bacterial infection was strongly suppressed in HcTRAF6-silenced mussels and that depletion of HcTRAF inhibited the elimination of A. hydrophila. All these findings together prove that HcTRAF6 functions as an efficient regulator in innate immune mechanisms against invading pathogens and the alloimmune mechanism after mantle implantation in H. cumingii.

Characterization of allograft inflammatory factor-1 in Hyriopsis cumingii and its expression in response to immune challenge and pearl sac formation.

The allograft inflammatory factor-1 (AIF-1) is one of the key factors associated with inflammatory response and immune defense. In the present study, we report the identification and characterization of AIF-1 from triangle sail mussel Hyriopsis cumingii (HcAIF-1). The full-length cDNA of HcAIF-1 consisted of a 5'-terminal untranslated region (UTR) of 80 bp, a 3'-UTR of 420 bp with a poly (A) tail, and an open reading frame of 444 bp encoding a polypeptide of 147 amino acids with two conserved EF-hand Ca2+-binding motifs. HcAIF-1 mRNA and protein were expressed in all examined tissues and showed higher mRNA expression levels were observed in immune tissues, especially hemocytes and mantle, and the highest protein expression level was in mantle. The expression level of HcAIF-1 mRNA was significantly upregulated in hemocytes 12-48 h after lipopolysaccharide challenge. After mantle tissue implantation, the expression level of this gene in pearl sac decreased significantly at 3-48 h (P < 0.01), and then was significantly upregulated at 96 h (P < 0.05) and recovered to the control level at 21-28 d. There was significant increase HcAIF-1 transcript abundance in hemocytes 96 h (P < 0.05) after mantle tissue implantation. The phagocytosis rate was significantly enhanced in hemocytes 3-24 h (P < 0.01) after the injection of recombinant HcAIF-1 protein. These findings suggest that HcAIF-1 is important in the underlying mechanism of the innate immune responses and pearl sac formation of H. cumingii.

A Novel Matrix Protein Hic31 from the Prismatic Layer of Hyriopsis Cumingii Displays a Collagen-Like Structure.

In this study, we clone and characterize a novel matrix protein, hic31, from the mantle of Hyriopsis cumingii. The amino acid composition of hic31 consists of a high proportion of Glycine residues (26.67%). Tissue expression detection by RT-PCR indicates that hic31 is expressed specifically at the mantle edge. In situ hybridization results reveals strong signals from the dorsal epithelial cells of the outer fold at the mantle edge, and weak signals from inner epithelial cells of the same fold, indicating that hic31 is a prismatic-layer matrix protein. Although BLASTP results identify no shared homology with other shell-matrix proteins or any other known proteins, the hic31 tertiary structure is similar to that of collagen I, alpha 1 and alpha 2. It has been well proved that collagen forms the basic organic frameworks in way of collagen fibrils and minerals present within or outside of these fibrils. Therefore, hic31 might be a framework-matrix protein involved in the prismatic-layer biomineralization. Besides, the gene expression of hic31 increase in the early stages of pearl sac development, indicating that hic31 may play important roles in biomineralization of the pearl prismatic layer.

Identification of housekeeping genes suitable for gene expression analysis in the pearl mussel, Hyriopsis cumingii, during biomineralization.

Quantitative real-time polymerase chain reaction is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcript levels across different samples and tissues. The freshwater pearl, Hyriopsis cumingii (Lea), is an important economic species cultured in China. To date, no reference genes for gene expression analysis in this species have been validated. This study aimed to compare the relative expression of seven housekeeping genes across different tissue types and in the mantle or pearl sac during three biomineralization processes: seasonal shell growth, shell healing and pearl-sac formation in H. cumingii. Three programs evaluated the expression stabilities of the seven genes: BestKeeper, geNorm and NormFinder. The beta actin gene (ACTB), commonly used as a housekeeping gene in many studies, was the least stable. The expressions of Ubiquitin (Ubi) and Ribosomal protein L18 (Rpl18) and Elongation factor 1-alpha (EF1α) were more stable than the remaining four genes. Therefore, we suggest that Ubi, Rpl18 and EF1α are suitable reference genes. The three selected reference genes are expected to facilitate analysis of gene expressions during shell or pearl formation in H. cumingii.

Cloning, differential tissue expression of a novel hcApo gene, and its correlation with total carotenoid content in purple and white inner-shell color pearl mussel Hyriopsis cumingii.

As a molecular carrier and storage protein, apolipoprotein (Apo) mediates the intracellular uptake of lipids, proteins, vitamins and carotenoids. In this study, we identified a novel Apo gene, designated hcApo, from the freshwater pearl mussel Hyriopsis cumingii. The complete hcApo cDNA consists of 4104 nucleotides with an open reading frame encoding 1155 amino acid residues. The hcApo protein contains a conserved lipoprotein N-terminal domain (LPD-N) that is a characteristic of the large lipid transfer protein (LLTP) superfamily. The hcApo mRNA is constitutively expressed in a wide range of tissues with the highest expression level in the liver. Moreover, differential expression analysis revealed that the hcApo gene is more highly expressed in the liver, kidney, mantle and gill of purple line mussels compared to white line mussels. In situ hybridization investigations of the precise expression site of hcApo mRNA in the mantle showed that hcApo mRNA is specifically expressed in the outer epithelial cells of the middle fold and the inner epithelial cells of the outer fold of the mantle, as well as throughout the outer epithelium of the outer fold and ventral mantle. Another very important finding is that significantly positive correlation existed between the hcApo gene expression level and the total carotenoid content in purple line mussels. These findings may provide a better understanding of the roles of hcApo in the molecular mechanisms of shell formation and coloring of H. cumingii.

Characterization of MMP-9 gene from grass carp (Ctenopharyngodon idella): an Aeromonas hydrophila-inducible factor in grass carp immune system.

Matrix metalloproteinase-9 (MMP-9) belongs to a family of zinc-dependent endopeptidases and is associated with vital inflammatory processes. Here, we isolated and characterized MMP-9 cDNA from grass carp (Ctenopharyngodon idella) (designated as CiMMP-9). The cDNA was 2880 bp long and encoded a putative protein of 675 amino acids, with a predicted molecular mass of 75.816 kDa and an isoelectric point (pI) of 5.25. CiMMP-9 contained all three classical MMP-9 family signatures. The mRNA of CiMMP-9 was constitutively expressed in all tested tissues of untreated grass carp, with the highest expression levels in the blood, trunk kidney, head kidney and spleen. CiMMP9 transcript was present in unfertilized eggs, which suggests that CiMMP9 transcription is maternally inherited. Fluorescent real-time quantitative RT-PCR was used to examine the expression of the CiMMP-9 gene in C. idella after being challenged with Aeromonas hydrophila. A clear time-dependent expression pattern of CiMMP-9 was found after the bacterial challenge, and mRNA expression reached a maximum level at 7 days post challenge. This indicates that MMP-9 is inducible and is involved in immune responses, thus suggesting that CiMMP-9 plays an important role in A. hydrophila-related diseases and in early embryonic development stages in C. idella.

Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii.

Perlucin is an important functional protein that regulates shell and pearl formation. In this study, we cloned the perlucin gene from the freshwater pearl mussel Hyriopsis cumingii, designated as Hcperlucin. The full-length cDNA transcribed from the Hcperlucin gene was 1460 bp long, encoding a putative signal peptide of 20 amino acids and a mature protein of 141 amino acids. The mature Hcperlucin peptide contained six conserved cysteine residues and a carbohydrate recognition domain, similar to other members of the C-type lectin families. In addition, a "QPS" and an invariant "WND" motif near the C-terminal region were also found, which are extremely important for polysaccharide recognition and calcium binding of lectins. The mRNA of Hcperlucin was constitutively expressed in all tested H. cumingii tissues, with the highest expression levels observed in the mantle, adductor, gill and hemocytes. In situ hybridization was used to detect the presence of Hcperlucin mRNA in the mantle, and the result showed that the mRNA was specifically expressed in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the biosynthesis of the nacreous layer of the shell. The significant Hcperlucin mRNA expression was detected on day 14 post shell damage and implantation, suggesting that the Hcperlucin might be an important gene in shell nacreous layer and pearl formation. The change of perlucin expression in pearl sac also confirmed that the mantle transplantation results in a new expression pattern of perlucin genes in pearl sac cells that are required for pearl biomineralization. These findings could help better understanding the function of perlucin in the shell and pearl formation.

Molecular cloning and copy number variation of a ferritin subunit (Fth1) and its association with growth in freshwater pearl mussel Hyriopsis cumingii.

Iron is one of the most important minor elements in the shells of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell growth. A novel ferritin subunit (Fth1) cDNA from the freshwater pearl mussel (Hyriopsis cumingii) was isolated and characterized. The complete cDNA contained 822 bp, with an open reading frame (ORF) of 525 bp, a 153 bp 5' untranslated region (UTR) and a 144 bp 3' UTR. The complete genomic DNA was 4125 bp, containing four exons and three introns. The ORF encoded a protein of 174 amino acids without a signal sequence. The deduced ferritin contained a highly conserved motif for the ferroxidase center comprising seven residues of a typical vertebrate heavy-chain ferritin. It contained one conserved iron associated residue (Try27) and iron-binding region signature 1 residues. The mRNA contained a 27 bp iron-responsive element with a typical stem-loop structure in the 5'-UTR position. Copy number variants (CNVs) of Fth1 in two populations (PY and JH) were detected using quantitative real-time PCR. Associations between CNVs and growth were also analyzed. The results showed that the copy number of the ferritin gene of in the diploid genome ranged from two to 12 in PY, and from two to six in JH. The copy number variation in PY was higher than that in JH. In terms of shell length, mussels with four copies of the ferritin gene grew faster than those with three copies (P<0.05), suggesting that CNVs in the ferritin gene are associated with growth in shell length and might be a useful molecular marker in selective breeding of H. cumingii.