Expression characteristics and in vitro antibacterial properties of C-type lysozyme in crucian carp infected with Aeromonas salmonicida.

Aeromonas salmonicida is an ancient fish pathogen. Lysozymes are important molecules in the innate immune system that fight bacterial infections. The expression characteristics of C-type lysozyme in crucian carp infected with A. salmonicida and its antibacterial effect against A. salmonicida had not been investigated. Thus, we used bioinformatics to analyze the gene and protein characteristics of C-type lysozymes in crucian carp. Changes in C-type lysozyme expression before and after crucian carp infection with A. salmonicida were detected, and the in vitro antibacterial effect of recombinant carp C-type lysozyme on A. salmonicida was validated. The results showed that the coding DNA sequence region of the lysozyme gene sequence was 438 bp long, encoding 145 amino acids and containing two conserved catalytic sites: Glu53 and Asp69. Phylogenetic analysis revealed that crucian carp C-type lysozymes clustered with Cyprinus carpio lysozyme C. After crucian carp were infected with A. salmonicida, the gene and protein expression of C-type lysozymes in the liver, spleen, kidney, and hindgut were significantly upregulated, with the liver showing the highest upregulation that was 15 times higher than that in the uninfected group. In addition, recombinant C-type lysozyme exhibited significant antibacterial activity against A. salmonicida, with an average inhibition zone radius of 0.92 cm when using 40 µg recombinant lysozyme. In conclusion, this study reveals the important role of C-type lysozymes in the innate immune response of crucian carp and provides a theoretical basis for preventing crucian carp infection with A. salmonicida.

Analysis of bone metabolic alterations linked with osteoporosis progression in type 2 diabetic db/db mice.

Type 2 diabetes (T2D) is a common chronic disease, characterized by persistent hyperglycemia and insulin resistance. This disorder is associated with decreased bone quality and an elevated risk of bone fractures. However, evidence on the relationship between systemic metabolic change and the development of type 2 diabetic osteoporosis (T2DOP) remains elusive. Herein, we investigate the changes of bone metabolites with bone loss in db/db mice (an animal model of T2DOP exhibited bone loss with age progression), and explore the potential metabolic mechanism underlying type 2 diabetes and osteoporosis. C57BKS male mice were distributed in four groups, consisting six mice in each group: 8w m/m, 24w m/m, 8w db/db and 24w db/db. Bone morphometric and biomechanical parameters of db/db mice were analyzed by micro-CT and materials tester, it was found that 24w db/db mice showed severe bone loss and decreased bone tissue hardness compared with misty/misty littermates. The tibia of misty/misty mice (8 weeks, 24 weeks) and db/db mice (8 weeks, 24 weeks) were screened for differential metabolites by UPLC-Orbitrap MS. Ninety-eight metabolites were identified (35 and 63 metabolites are associated with early staged and late staged, respectively), consisting of amino acids, fatty acyls, and nucleotides. Notably, fatty acyls (such as 18-HEPE, 16(17)-EpDPE, arachidonic acid) and glycerophospholipids (such as phosphocholines (PC) (O-10:1(9E)/0:0), PC (O-16:1(9E)/0:0) [U] and phosphatidylethanolamines (PE) (P-16:0/0:0)) were significantly increased, and metabolites of amino acid pathway (such as l-glutamine, proline, phenylalanine) showed a downregulation trend. Dysregulation of lipid and glutathione pathways is the major contributor to progression of T2DOP in C57BKS mice.

Oleanolic acid derivative alleviates cardiac fibrosis through inhibiting PTP1B activity and regulating AMPK/TGF-ß/Smads pathway.

Cardiac fibrosis (CF) in response to persistent exogenous stimuli or myocardial injury results in cardiovascular diseases (CVDs). Protein tyrosine phosphatase 1B (PTP1B) can promote collagen deposition through regulating AMPK/TGF-ß/Smads signaling pathway, and PTP1B knockout improves cardiac dysfunction against overload-induced heart failure. Oleanolic acid (OA) has been proven to be an inhibitor of PTP1B, and its anti-cardiac remodeling effects have been validated in different mouse models. To improve the bioactivity of OA and to clarify whether OA derivatives with stronger inhibition of PTP1B activity have greater prevention of cardiac remodeling than OA, four new OA derivatives were synthesized and among them, we found that compound B had better effects than OA in inhibiting cardiac fibrosis both in vivo in the isoproterenol (ISO)-induced mouse cardiac fibrosis and in vitro in the TGF-ß/ISO-induced 3T3 cells. Combining with the results of molecular docking, surface plasmon resonance and PTP1B activity assay, we reported that OA and compound B directly bound to PTP1B and inhibited its activity, and that compound B showed comparable binding capability but stronger inhibitory effect on PTP1B activity than OA. Moreover, compound B presented much greater effects on AMPK activation and TGF-ß/Smads inhibition than OA. Taken together, OA derivative compound B more significantly alleviated cardiac fibrosis than OA through much greater inhibition of PTP1B activity and thus much stronger regulation of AMPK/TGF-ß/Smads signaling pathway.

Inflammatory Milieu Related to Dysbiotic Gut Microbiota Promotes Tumorigenesis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an invasive primary liver cancer caused by multiple pathogenic factors and is a significant global health concern. With few effective therapeutic options, HCC is a heterogeneous carcinoma that typically arises in an inflammatory environment. Recent studies have suggested that dysbiotic gut microbiota is involved in hepatocarcinogenesis via multiple mechanisms. In this review, we discuss the effects of gut microbiota, microbial components, and microbiota-derived metabolites on the promotion and progression of HCC by feeding a persistent inflammatory milieu. In addition, we discuss the potential therapeutic modalities for HCC targeting the inflammatory status induced by gut microbiota. A better understanding of the correlation between the inflammatory milieu and gut microbiota in HCC may be beneficial for developing new therapeutic strategies and managing the disease.

Antihepatoma peptide, scolopentide, derived from the centipede scolopendra subspinipes mutilans.

BACKGROUND: Centipedes have been used to treat tumors for hundreds of years in China. However, current studies focus on antimicrobial and anticoagulation agents rather than tumors. The molecular identities of antihepatoma bioactive components in centipedes have not yet been extensively investigated. It is a challenge to isolate and characterize the effective components of centipedes due to limited peptide purification technologies for animal-derived medicines. AIM: To purify, characterize, and synthesize the bioactive components with the strongest antihepatoma activity from centipedes and determine the antihepatoma mechanism. METHODS: An antihepatoma peptide (scolopentide) was isolated and identified from the centipede scolopendra subspinipes mutilans using a combination of enzymatic hydrolysis, a Sephadex G-25 column, and two steps of high-performance liquid chromatography (HPLC). Additionally, the CCK8 assay was used to select the extracted fraction with the strongest antihepatoma activity. The molecular weight of the extracted scolopentide was characterized by quadrupole time of flight mass spectrometry (QTOF MS), and the sequence was matched by using the Mascot search engine. Based on the sequence and molecular weight, scolopentide was synthesized using solid-phase peptide synthesis methods. The synthetic scolopentide was confirmed by MS and HPLC. The antineoplastic effect of extracted scolopentide was confirmed by CCK8 assay and morphological changes again in vitro. The antihepatoma effect of synthetic scolopentide was assessed by the CCK8 assay and Hoechst staining in vitro and tumor volume and tumor weight in vivo. In the tumor xenograft experiments, qualified model mice (male 5-week-old BALB/c nude mice) were randomly divided into 2 groups (n = 6): The scolopentide group (0.15 mL/d, via intraperitoneal injection of synthetic scolopentide, 500 mg/kg/d) and the vehicle group (0.15 mL/d, via intraperitoneal injection of normal saline). The mice were euthanized by cervical dislocation after 14 d of continuous treatment. Mechanistically, flow cytometry was conducted to evaluate the apoptosis rate of HepG2 cells after treatment with extracted scolopentide in vitro. A Hoechst staining assay was also used to observe apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. CCK8 assays and morphological changes were used to compare the cytotoxicity of synthetic scolopentide to liver cancer cells and normal liver cells in vitro. Molecular docking was performed to clarify whether scolopentide tightly bound to death receptor 4 (DR4) and DR5. qRT-PCR was used to measure the mRNA expression of DR4, DR5, fas-associated death domain protein (FADD), Caspase-8, Caspase-3, cytochrome c (Cyto-C), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), x-chromosome linked inhibitor-of-apoptosis protein and Cellular fas-associated death domain-like interleukin-1ß converting enzyme inhibitory protein in hepatocarcinoma subcutaneous xenograft tumors from mice. Western blot assays were used to measure the protein expression of DR4, DR5, FADD, Caspase-8, Caspase-3, and Cyto-C in the tumor tissues. The reactive oxygen species (ROS) of tumor tissues were tested. RESULTS: In the process of purification, characterization and synthesis of scolopentide, the optimal enzymatic hydrolysis conditions (extract ratio: 5.86%, IC50: 0.310 mg/mL) were as follows: Trypsin at 0.1 g (300 U/g, centipede-trypsin ratio of 20:1), enzymolysis temperature of 46 °C, and enzymolysis time of 4 h, which was superior to freeze-thawing with liquid nitrogen (IC50: 3.07 mg/mL). A peptide with the strongest antihepatoma activity (scolopentide) was further purified through a Sephadex G-25 column (obtained A2) and two steps of HPLC (obtained B5 and C3). The molecular weight of the extracted scolopentide was 1018.997 Da, and the peptide sequence was RAQNHYCK, as characterized by QTOF MS and Mascot. Scolopentide was synthesized in vitro with a qualified molecular weight (1018.8 Da) and purity (98.014%), which was characterized by MS and HPLC. Extracted scolopentide still had an antineoplastic effect in vitro, which inhibited the proliferation of Eca-109 (IC50: 76.27 µg/mL), HepG2 (IC50: 22.06 µg/mL), and A549 (IC50: 35.13 µg/mL) cells, especially HepG2 cells. Synthetic scolopentide inhibited the proliferation of HepG2 cells (treated 6, 12, and 24 h) in a concentration-dependent manner in vitro, and the inhibitory effects were the strongest at 12 h (IC50: 208.11 µg/mL). Synthetic scolopentide also inhibited the tumor volume (Vehicle vs Scolopentide, P = 0.0003) and weight (Vehicle vs Scolopentide, P = 0.0022) in the tumor xenograft experiment. Mechanistically, flow cytometry suggested that the apoptosis ratios of HepG2 cells after treatment with extracted scolopentide were 5.01% (0 µg/mL), 12.13% (10 µg/mL), 16.52% (20 µg/mL), and 23.20% (40 µg/mL). Hoechst staining revealed apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. The CCK8 assay and morphological changes indicated that synthetic scolopentide was cytotoxic and was significantly stronger in HepG2 cells than in L02 cells. Molecular docking suggested that scolopentide tightly bound to DR4 and DR5, and the binding free energies were-10.4 kcal/mol and-7.1 kcal/mol, respectively. In subcutaneous xenograft tumors from mice, quantitative real-time polymerase chain reaction and western blotting suggested that scolopentide activated DR4 and DR5 and induced apoptosis in SMMC-7721 Liver cancer cells by promoting the expression of FADD, caspase-8 and caspase-3 through a mitochondria-independent pathway. CONCLUSION: Scolopentide, an antihepatoma peptide purified from centipedes, may inspire new antihepatoma agents. Scolopentide activates DR4 and DR5 and induces apoptosis in liver cancer cells through a mitochondria-independent pathway.

The Nucleus Accumbens CRH-CRHR1 System Mediates Early-Life Stress-Induced Sleep Disturbance and Dendritic Atrophy in the Adult Mouse / 神经科学通报·英文版

Adverse experiences in early life have long-lasting negative impacts on behavior and the brain in adulthood, one of which is sleep disturbance. As the corticotropin-releasing hormone (CRH)-corticotropin-releasing hormone receptor 1 (CRHR1) system and nucleus accumbens (NAc) play important roles in both stress responses and sleep-wake regulation, in this study we investigated whether the NAc CRH-CRHR1 system mediates early-life stress-induced abnormalities in sleep-wake behavior in adult mice. Using the limited nesting and bedding material paradigm from postnatal days 2 to 9, we found that early-life stress disrupted sleep-wake behaviors during adulthood, including increased wakefulness and decreased non-rapid eye movement (NREM) sleep time during the dark period and increased rapid eye movement (REM) sleep time during the light period. The stress-induced sleep disturbances were accompanied by dendritic atrophy in the NAc and both were largely reversed by daily systemic administration of the CRHR1 antagonist antalarmin during stress exposure. Importantly, Crh overexpression in the NAc reproduced the effects of early-life stress on sleep-wake behavior and NAc morphology, whereas NAc Crhr1 knockdown reversed these effects (including increased wakefulness and reduced NREM sleep in the dark period and NAc dendritic atrophy). Together, our findings demonstrate the negative influence of early-life stress on sleep architecture and the structural plasticity of the NAc, and highlight the critical role of the NAc CRH-CRHR1 system in modulating these negative outcomes evoked by early-life stress.

Research progress on the immunomodulatory effects and mechanisms of trace amine-associated receptor 1 / 生理学报

Trace amines are endogenous molecules distributed in the central nervous system and peripheral tissues that resemble common biogenic amines in terms of subcellular localization, chemical structure, and metabolism. Trace amine-associated receptor (TAAR) is a kind of evolutionarily conserved G-protein-coupled receptors in vertebrates, in which TAAR1 is a functional regulator of monoamine transmitters such as dopamine and serotonin. TAAR1 is widely considered as a potential therapeutic target for schizophrenia, depression and drug addiction. Moreover, TAAR1 is also expressed in peripheral tissues. The homeostasis imbalance of trace aminergic system can induce over-activation of peripheral immune system and central immune inflammatory response. TAAR1 modulators are becoming potential emerging drugs for the treatment of immune-related illnesses, because they may play a major role in the activation or modulation of immune response.

Dysbiosis: The first hit for digestive system cancer.

Gastrointestinal cancer may be associated with dysbiosis, which is characterized by an alteration of the gut microbiota. Understanding the role of gut microbiota in the development of gastrointestinal cancer is useful for cancer prevention and gut microbiota-based therapy. However, the potential role of dysbiosis in the onset of tumorigenesis is not fully understood. While accumulating evidence has demonstrated the presence of dysbiosis in the intestinal microbiota of both healthy individuals and patients with various digestive system diseases, severe dysbiosis is often present in patients with digestive system cancer. Importantly, specific bacteria have been isolated from the fecal samples of these patients. Thus, the association between dysbiosis and the development of digestive system cancer cannot be ignored. A new model describing this relationship must be established. In this review, we postulate that dysbiosis serves as the first hit for the development of digestive system cancer. Dysbiosis-induced alterations, including inflammation, aberrant immune response, bacteria-produced genotoxins, and cellular stress response associated with genetic, epigenetic, and/or neoplastic changes, are second hits that speed carcinogenesis. This review explains the mechanisms for these four pathways and discusses gut microbiota-based therapies. The content included in this review will shed light on gut microbiota-based strategies for cancer prevention and therapy.

A specific reverse complement sequence for distinguishing Brucella canis from other Brucella species.

Canine brucellosis is primarily caused by Brucella canis, but other Brucella species can also cause the disease. Identifying sequences specific to B. canis and establishing PCR assays that can distinguish between B. canis and other Brucella species is essential to determine the etiology of canine brucellosis and the source of infection and to achieve effective control. We analyzed the gaps and SNPs of genomes I and II from B. canis strain RM6/66 and B. melitensis strain 16M using the Mauve genome alignment software, and the specificity of each of these differential regions was analyzed by BLAST. A 132 bp specific sequence was found between the DK60_915 (glycosyl hydrolase 108 family protein) and DK60_917 (aldose 1-epimerase) loci in B. canis chromosome 1. Further comparative analysis revealed that this is a reverse complement sequence between B. canis and other Brucella species. Then, three primers were designed based on the sequence that could detect B. canis with a 310 bp amplification product or other Brucella species with a 413 bp product. The PCR based on these primers had reasonable specificity and a sensitivity of 100 copies of Brucella DNA. The detection results for the blood samples of the aborted dogs showed a favorable accordance with the Bruce-ladder multiplex PCR assay. In conclusion, we found a specific reverse complement sequence between B. canis and other Brucella and developed a PCR method that allows a more comprehensive identification of the pathogen involved in canine brucellosis. These findings provide an effective means for preventing and controlling brucellosis.

Pan-cancer analysis of oncogenic TNFAIP2 identifying its prognostic value and immunological function in acute myeloid leukemia.

BACKGROUND: Tumor necrosis factor alpha-induced protein 2 (TNFAIP2), a TNFα-inducible gene, appears to participate in inflammation, immune response, hematopoiesis, and carcinogenesis. However, the potential role of TNFAIP2 in the development of acute myeloid leukemia (AML) remains unknow yet. Therefore, we aimed to study the biological role of TNFAIP2 in leukemogenesis. METHODS: TNFAIP2 mRNA level, prognostic value, co-expressed genes, differentially expressed genes, DNA methylation, and functional enrichment analysis in AML patients were explored via multiple public databases, including UALCAN, GTEx portal, Timer 2.0, LinkedOmics, SMART, MethSurv, Metascape, GSEA and String databases. Data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and Beat AML database were used to determine the associations between TNFAIP2 expression and various clinical or genetic parameters of AML patients. Moreover, the biological functions of TNFAIP2 in AML were investigated through in vitro experiments. RESULTS: By large-scale data mining, our study indicated that TNFAIP2 was differentially expressed across different normal and tumor tissues. TNFAIP2 expression was significantly increased in AML, particularly in French-American-British (FAB) classification M4/M5 patients, compared with corresponding control tissues. Overexpression of TNFAIP2 was an independent poor prognostic factor of overall survival (OS) and was associated with unfavorable cytogenetic risk and gene mutations in AML patients. DNA hypermethylation of TNFAIP2 at gene body linked to upregulation of TNFAIP2 and inferior OS in AML. Functional enrichment analysis indicated immunomodulation function and inflammation response of TNFAIP2 in leukemogenesis. Finally, the suppression of TNFAIP resulted in inhibition of proliferation by altering cell-cycle progression and increase of cell death by promoting early and late apoptosis in THP-1 and U937AML cells. CONCLUSION: Collectively, the oncogenic TNFAIP2 can function as a novel biomarker and prognostic factor in AML patients. The immunoregulation function of TNFAIP2 warrants further validation in AML.

[Mechanism of combined treatment of rhein and emodin in Rhubarb for ulcerative colitis].

This study aimed to explore the efficacy and mechanism of combined rhein and emodin in the treatment of ulcerative colitis(UC) from the aspects of network pharmacology, animal inflammation improvement and molecular mechanism. Network pharmacology predicted that combined rhein and emodin acted on 52 potential targets, mainly participating in signaling pathways such as cancer, PI3 K/AKT, microRNAs in cancer and apoptosis. PI3 K/AKT signaling pathway has been reported to be closely related to UC, and the optimal candidate pathway for combined therapy. The UC mice model was established by dextran sodium sulfate, and then the modeled mice were randomly divided into control group, model group, rhein group, emodin group, rhein+emodin group and sulfasalazine group. After administration, compared with the conditions in model group, body weight, disease activity index(DAI) score, colon length, TNF-α, IL-6, IL-1ß and myeloperoxidase(MPO) of mice in rhein+emodin group were improved(P<0.01); colonic mucosal injury was significantly reduced; the expression of p-PI3 K/PI3 K and p-AKT/AKT proteins were down-regulated(P<0.01). All the above indices were better than those in the rhein/emodin group alone. The Jin's Q-values of the effect of combined rhein and emodin on colon length, TNF-α, IL-6, IL-1ß, MPO, p-PI3 K/PI3 K and p-AKT/AKT were all greater than 1.15, which indicated that there was obvious synergistic effect between rhein and emodin. In all, rhein and emodin have synergistic effect in the treatment of UC, and the mechanism may be related to the inhibition of PI3 K/AKT signaling pathway and the down-regulation of proinflammatory factors. They are the new components in the treatment of UC, which is worthy of attention.

[Comparative study on intestinal absorption kinetics of main active components in Sini Decoction and its separated recipes].

This paper aims to study the difference in the intestinal absorption kinetics of main active components of Sini decoction and its separated recipes and explain the scientificity and rationality of the compatibility of Sini Decoction. A in situ intestinal perfusion rat model was established to evaluate the differences in the absorption of benzoylmesaconine, benzoylaconine, benzoylhypacoitine, mesaconitine, hypaconitine, glycyrrhizic acid, liquiritin and 6-gingerol from Sini Decoction and its separated recipes in the duodenum, jejunum and ileum by high performance liquid chromatography(HPLC). The results indicated that the Sini Decoction group was superior to the Aconiti Lateralis Radix Praeparata group in terms of absorption degree and rate for aconitum alkaloids. The absorption of benzoylmesaconine and hypaconitine in the duodenum, jejunum and ileum was faster and stronger in the Sini Decoction group(P<0.05). The absorption degree of glycyrrhizic acid in the duodenum was significantly higher in the Sini Decoction group than in the Glycyrrhizae Radix et Rhizoma group and the Glycyrrhizae Radix et Rhizoma-Zingiberis Rhizoma group(P<0.05). The absorption rate and degree of 6-gingerol in the ileum in the Sini Decoction group were significantly higher than those in the Zingiberis Rhizoma group(P<0.05). In short, Zingiberis Rhizoma and Glycyrrhizae Radix et Rhizoma can promote the absorption of aconitum alkaloids in different intestinal segments, which reflects the scientific composition of Sini Decoction.

Anti-melanogenesis effect from Wampee fruit pectin via α-MSH/TRY pathway in A375 cells.

BACKGROUND: Polysaccharides from wampee have been reported to process various biological activities, while the relationship between structure and bioactivities has been barely addressed. Pectin, an abundant water-soluble polysaccharide in wampee, showed significant antioxidant activity, which was associated with the anti-melanogenic activity. Therefore, this study investigated the physicochemical characteristics and the anti-melanogenesis effect of pectin extracted from wampee fruit in A375 cells. METHODS: The physicochemical characterization of pectin from wampee fruit was investigated by gel chromatography (GCP), FT-IR spectroscopy, and NMR spectroscopy methods. The anti-melanogenesis effects and mechanism were evaluated by mushroom tyrosine enzyme and human melanin cell model in vitro. RESULTS: The results showed that a molecular weight of 5.271 × 105 Da wampee fruit pectin (WFP) were mainly composed of mannose (Man), ribose (Rib), rhamnose (Rha), glucuronic acid (Glc A), glucose (Glc), galacturonic acid (Gal A), galactose (Gal), and arabinose (Ara), which linked with →4)-ß-D-Galp-(1 → units. The current study revealed that WFP could significantly suppress mushroom TRY activity in vitro. Furtherly, WFP significantly reduced intracellular and extracellular melanin formation in A375 melanoma cells depending on the presence of alpha-melanocyte stimulating hormone (α-MSH). TRY activity was only inhibited in α-MSH treated A375 cells. Western blot analysis demonstrated that WFP reverse α-MSH induced melanogenesis in A375 melanoma cells, including in down-regulated TRY, TYRP-1, TYRP-2, MITF and CREB expressions. CONCLUSION: These results indicated that WFP could inhibit α-MSH induced melanogenesis in A375 melanoma cells via α-MSH/TRY pathway. In conclusion, these data provided a new perspective to annotate WFP anti-melanogenesis activity mechanism.

Abnormal Epigenetic Regulations in the Immunocytes of Sjögren's Syndrome Patients and Therapeutic Potentials.

Sjögren's syndrome (SjS), characterized by keratoconjunctivitis sicca and dry mouth, is a common autoimmune disease, especially in middle-aged women. The immunopathogenesis of SjS is caused by the sequential infiltration of T and B cells into exocrine glands, including salivary and lacrimal glands. Effector cytokines produced by these immunocytes, such as interferons (IFNs), IL-17, IL-22, IL-21, IL-4, TNF-α, BAFF and APRIL, play critical roles in promoting autoimmune responses and inducing tissue damages. Epigenetic regulations, including DNA methylation, histone modification and non-coding RNAs, have recently been comprehensively studied during the activation of various immunocytes. The deficiency of key epigenetic enzymes usually leads to aberrant immune activation. Epigenetic modifications in T and B cells are usually found to be altered during the immunopathogenesis of SjS, and they are closely correlated with autoimmune responses. In particular, the important role of methylation in activating IFN pathways during SjS progression has been revealed. Thus, according to the involvement of epigenetic regulations in SjS, target therapies to reverse the altered epigenetic modifications in auto-responsive T and B cells are worthy of being considered as a potential therapeutic strategy for SjS.

Structural characterization of a mannoglucan polysaccharide from Dendrobium huoshanense and evaluation of its osteogenesis promotion activities.

Dendrobium huoshanense, a valuable traditional Chinese herb, is widely used to prolong life in China. Our study aims to characterize the structure and osteogenesis-promotion effects of a heteropolysaccharide component isolated from Dendrobium huoshanense (DHPW1). The structure of DHPW1 was characterized using gas chromatography-mass spectrometry and nuclear magnetic resonance, while its osteogenic activity was evaluated using MG-63 cells and zebrafish skulls. The results showed that the molecular weight of DHPW1 was 230 kDa and it was mainly composed of mannose and glucose. In addition, the DHPW1 backbone consisted of (1 â†’ 4)-linked-ß-D-Mannopyranosyl and (1 â†’ 4)-linked-ß-d-Glucopyranosyl. Furthermore, DHPW1 significantly increased ALP activity and mineralized nodule formation in MG-63 cells. DHPW1 in zebrafish skull models significantly enhanced the relative fluorescence intensity of bone mass and increased the degree of bone mineralization. These results suggested that the DHPW1 component in D. huoshanense has potential to promote osteogenesis.

Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin pathway.

BACKGROUND: The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin (PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills (XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma (HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHP-associated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway. AIM: To confirm the effect of XHP on HCC and the possible mechanisms involved. METHODS: The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). Cell-based experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP (0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay. Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively. Third, Western blotting and RT-qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway. Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed. RESULTS: The following 12 compounds were identified in XHP using high-resolution mass spectrometry: Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-ß-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-ß-boswellic acid, 5ß-androstane-3,17-dione, and 3-acetyl-11-keto-ß-boswellic acid. The cell viability assay results showed that treatment with 0.625 mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose- and time-dependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract (0.625 mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins (e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights. CONCLUSION: XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3. Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.

[Effects of simulated rainfall enhancement on sediment CO2 flux in dry lakebed of Barkol Lake, China]. / 模拟增雨对巴里坤湖干涸湖底沉积物CO2通量的影响.

Understanding the responses of lake sediment carbon process to climate change is an important part of a comprehensive understanding of lake carbon budget. To explore the effects of future rainfall increase on sediment carbon flux, undisturbed sediment samples were collected from the bottom of dry lake Barikun in Hami, Xinjiang for the incubation experiment. Based on the increase rate of precipitation (4 mm·10 a-1) and the distribution characteristics of rainfall in the plant growing season in Hami, Xinjiang since 1960, five rainfall treatments were set (86 mm, T0; 94 mm, T1; 102 mm, T2; 110 mm, T3; 126 mm, T4) based on the rainfall in growing season of 2016 (86 mm). We analyzed the effects of rainfall increase on sediment CO2 flux. Results showed that compared with that before rainfall, the sediment CO2 flux increased after 1 day of rainfall in the study area. Compared with that during May to July, the CO2 flux of sediments in August to October decreased. There was no variation of CO2 accumulative emission among the T0-T3 treatments from May to October. However, the average CO2 emission rate under the T3 treatment (0.22 µmol·m-2·s-1) was significantly higher than that under the T4 treatment (0.14 µmol·m-2·s-1). All treatments showed CO2 sink at the first day of rainfall (1 d), with T4 treatment (-0.13 µmol·m-2·s-1) having the highest "carbon sink" capacity. After 1 day, the CO2 sink converted to CO2 source under the five rainfall treatments, with the CO2 emission rate under T3 treatment (0.34 µmol·m-2·s-1) being significantly higher than those under other treatments. Compared with May, the CO2 emission fluxes of T2-T4 treatments were significantly higher than those at the time from August to October. Under the condition with relatively stable temperature, the CO2 flux of sediments was significantly correlated with the sediment moisture and air humidity. In the next 60 years, the continuous increase of future rainfall may be an important factor promoting CO2 emission from lake sediment in arid regions, and thus affecting global warming.

Preliminary study on differentially expressed proteins in a mouse model of secondary alveolar echinococcosis based on data independent acquisition proteomics / 中国血吸虫病防治杂志

Objective To identify the differentially expressed proteins in different liver tissues in the mouse model of alveolar echinococcosis using high-resolution mass spectrometry with data independent acquisition (DIA), and to identify the key proteins contributing to the pathogenesis of alveolar echinococcosis. Methods Protoscoleces were isolated from Microtus fuscus with alveolar echinococcosis and the experimental model of alveolar echinococcosis was established in female Kunming mice aged 6 to 8 weeks by infection with Echinococcus multilocularis protoscoleces. Mice were divided into the experimental and control groups, and animals in the experimental group was injected with approximately 3 000 protoscoleces, while mice in the control group were injected with the same volume of physiological saline. Mouse liver specimens were sampled from both groups one year post-infection and subjected to pathological examinations. In addition, the lesions (the lesion group) and peri-lesion specimens (the peri-lesion group) were sampled from the liver of mice in the experimental group and the normal liver specimens (the normal group) were sampled from mice in the control group for DIA proteomics analysis, and the differentially expressed proteins were subjected to bioinformatics analysis. Results A total of 1 020 differentially expressed proteins were identified between the lesion group and the normal group, including 671 up-regulated proteins and 349 down-regulated proteins, and 495 differentially expressed proteins were identified between the peri-lesion group and the normal group, including 327 up-regulated proteins and 168 down-regulated proteins. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these differentially expressed proteins were involved in peroxisome, peroxisome proliferator-activated receptor (PPAR) and fatty acid degradation pathways, and the peroxisome and PPAR signaling pathways were found to correlate with liver injury. Several differentially expressed proteins that may contribute to the pathogenesis of alveolar echinococcosis were identified in these two pathways, including fatty acid binding protein 1 (Fabp1), Acyl-CoA synthetase long chain family member 1 (Acsl1), Acyl-CoA oxidase 1 (Acox1), Enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (Ehhadh) and Acetyl-Coenzyme A acyltransferase 1B (Acaa1b), which were down-regulated in mice in the experimental group. Conclusion A large number of differentially expressed proteins are identified in the liver of the mouse model of alveolar echinococcosis, and Fabp1, Acsl1, Acox1, Ehhadh and Acaa1b may contribute to the pathogenesis of alveolar echinococcosis.

Preliminary study on differentially expressed proteins in a mouse model of secondary cystic echinococcosis based on data independent acquisition proteomics / 中国血吸虫病防治杂志

Objective To identify the differentially expressed proteins in different liver tissues in the mouse model of cystic echinococcosis (CE), so as to provide insights into the research and development of therapeutic drugs targeting CE. Methods Female Kunming mice at ages of 6 to 8 weeks were randomly assigned into the CE group and the control group. Mice in the CE group were intraperitoneally infected with 2 000 Echinococcus multilocularis protoscoleces, while mice in the control group were injected with the same volume of physiological saline. All mice in both groups were sacrificed after breeding for 350 d, and the lesions (the lesion group) and peri-lesion specimens (the peri-lesion group) were sampled from the liver of mice in the CE group and the normal liver specimens (the normal group) were sampled from mice in the control group for data independent acquisition (DIA) proteomics analysis, and the differentially expressed proteins were subjected to Gene Ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Results A total of 26 differentially expressed proteins were identified between the lesion group and the normal group and between the peri-lesion group and the normal group, including 8 up-regulated proteins and 18 down-regulated proteins. GO term enrichment analysis showed that these differentially expressed proteins were predominantly enriched in endoplasmic reticulum membrane (biological components), oxidoreductase activity (molecular function) and oxoacid metabolic process and monocarboxylic acid metabolic process (biological processes). KEGG pathway enrichment analysis revealed that the differentially expressed protein Acyl-CoA oxidase 1 (Acox1), which contributed to primary bile acid biosynthesis during the fatty acid oxidation, was involved in peroxisome signaling pathway, and the differentially expressed protein fatty acid binding protein 1 (Fabp1), which contributed to fatty acid transport, was involved in the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Conclusion Differentially expressed proteins are identified in the liver specimens between mouse models of CE and normal mice, and some differentially expressed proteins may serve as potential drug targets for CE.

Niuhuang (Bovis Calculus)-Shexiang (Moschus) combination induces apoptosis and inhibits proliferation in hepatocellular carcinoma via PI3K/AKT/mTOR pathway / 数字中医药（英文）

@#Objective To investigate the effects of Niuhuang (Bovis Calculus, BC) and Shexiang (Moschus) (BC-Moschus) on human hepatocellular carcinoma (HCC) cells SMMC-7721 and a nude mouse model of subcutaneous xenografts, and to explore its anti-HCC mechanism. Methods The BC-Moschus combination was applied to two liver cancer models in vivo and in vitro. SMMC-7721 was divided into the BC-Moschus group and the control group, and different doses (rude drug dosage 0.625, 1.25, 2.5, and 5 mg/mL) of BC-Moschus extract were used for the intervention. The proliferation ability of HCC cells was detected using the Cell Counting Kit-8 (CCK-8) assay, and the migration ability was detected by a wound healing assay. A subcutaneous xenograft model was prepared using nude mice with human HCC. Specific pathogen-free-grade BALB/c nude mice (5-week-old) were randomly divided into the following groups (n = 6 per group): control (0.9% physiological saline 0.2 mL/d), BC-Moschus [BC 45.5 mg/(kg·d)+ Moschus 13 mg/(kg·d)], and cisplatin (DDP, intraperitoneal injection 5 mg/kg per week) groups. All groups were administered for 14 d. The volume and mass of the subcutaneous xenografts in nude mice were observed. The expression levels of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, apoptosis-associated factor p70 S6 Kinase (S6K), Bax, Bcl-2, caspase-3, and caspase-9 in nude mice subcutaneous xenografts were measured by real-time quantitative PCR (RT-qPCR) and Western blot. Terminal Deoxynucleotidy Transferase-Mediated dUTP Nick-End Labeling (TUNEL) was used for quantitative analysis of apoptotic cells. Results The CCK-8 assay demonstrated that the BC-Moschus combination inhibited HCC cell proliferation in a superior manner to the use of BC and Moschus alone, and the inhibition effect was dose- and time-dependent (P < 0.01). The wound healing assay showed that the BC-Moschus combination inhibited HCC cell migration (P < 0.01). In the subcutaneous xenograft model of nude mice with human HCC, we found that the tumor volume and weight of the BC-Moschus group were lower than those of the control group (P < 0.01). The levels of the PI3K/AKT/mTOR signaling pathway and S6K protein in the BC-Moschus and DDP groups were significantly decreased (P < 0.01). The expression level of the anti-apoptotic gene Bcl-2 was downregulated (P < 0.05), and the expression of the pro-apoptotic gene Bax and apoptosis-related factors caspase-3 and caspase-9 were significantly upregulated (P < 0.01). The TUNEL assays further confirmed that the combination of the BC-Moschuas could promote HCC (P < 0.01). Conclusion The BC-Moschus combination inhibited the proliferation and migration ability of HCC cells SMMC-7721 and effectively inhibited the growth of subcutaneous xenografts in nude mice. The mechanism may be closely related to the downregulation of the PI3K/AKT/mTOR pathway, regulation of apoptosis-related protein caspase-3, caspase-9, Bcl-2, and Bax expression, and promotion of apoptosis.