Aqueous extract of Sargentodoxa cuneata alleviates ulcerative colitis and its associated liver injuries in mice through the modulation of intestinal flora and related metabolites.

Background: Ulcerative colitis (UC) is a refractory disease worldwide. Liver injury can be found clinically with UC, and now, it is found that gut dysbiosis is an important mechanism in the pathogenesis of UC. Sargentodoxa cuneata has been used as a traditional Chinese medicine and is commonly used clinically for the treatment of UC. The main objective of this study was to investigate the intrinsic mechanisms of Sargentodoxa cuneata in the treatment of UC and its associated liver injuries from the perspective of intestinal flora and related metabolites. Methods: Ultra-performance liquid chromatography-mass spectrometry was used to identify the components in the aqueous extract of Sargentodoxa cuneata (AESc). Mice with UC induced by dextran sulfate sodium were used to study the effects of AESc on UC and its associated liver injuries. Furthermore, 16S rRNA gene sequencing and analysis were performed on intestinal contents, and correlation analysis of intestinal flora with short-chain fatty acids (SCFAs) and organic acids was performed. Results: A total of 114 compounds were identified in AESc. AESc improved disease activity index scores, liver index, and colon length in mice with UC and had a good protective effect on intestine and liver injuries. Moreover, the administration of AESc regulated gut microbiota dysbiosis and the levels of a few SCFAs and organic acids in mice with UC. In addition, the correlation analysis results showed that the Megamonas and Bifidobacterium were the key intestinal flora related to the levels of differential SCFAs and organic acids in mice with UC after AESc intervention. Conclusion: AESc has a good protective effect on UC and UC related liver injuries. Modulation of the intestinal flora and its metabolites (SCFAs and a few organic acids) is an important pathway for AESc in the treatment of UC and also provides a rationale for the clinical use of Sargentodoxa cuneata in the treatment of UC.

Anti-Ulcerative Colitis Effects and Active Ingredients in Ethyl Acetate Extract from Decoction of Sargentodoxa cuneata.

Ulcerative colitis (UC) is an intractable disease prevalent worldwide. While ethyl acetate extract from decoction of Sargentodoxa cuneata (EAdSc) has potential anti-inflammatory activity, its effects on UC remain unknown. In this study, the constituent compounds discussed in the literature and identified by gas chromatography and mass spectrometry (GC-MS) were collected, and the blood-soluble components of EAdSc were identified by liquid chromatography-mass spectrometry. The network pharmacology analysis and molecular docking analysis were performed to explore the potential underlying mechanism and active ingredients of EAdSc against UC. Furthermore, mice with dextran sulfate sodium (DSS)-induced UC were used to study the therapeutic effects and validate the mechanism of EAdSc against UC. A total of 53 compounds from EAdSc were identified in the literature and by GC-MS, and 22 blood-soluble EAdSc components were recognized. Network pharmacology analysis revealed that multiple inflammatory signaling pathways are involved in EAdSc's anti-UC activity. Furthermore, molecular docking analysis showed that the eleutheroside A, liriodendrin, epicatechin, 2-methoxy-4-vinylphenol, catechin, androsin, coumaroyltyramine, and catechol may be active against UC through the TLR4/NF-κB/NLRP3 pathway. EAdSc reduced the disease activity, macroscopic colon damage, and histological damage indices, as well as inhibiting DSS-induced spleen enlargement and colon shortening. In addition, EAdSc decreased the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and IL-17, as well as the expression of TLR4, NF-κB p65, NLRP3, and Caspase-1 mRNA in colon tissues. These results provide insights into the anti-UC effects and underlying mechanisms of EAdSc and help elucidate the active ingredients of EAdSc in the treatment of UC.

Lupenone improves type 2 diabetic nephropathy by regulating NF-κB pathway-mediated inflammation and TGF-ß1/Smad/CTGF-associated fibrosis.

BACKGROUND: Type 2 diabetic nephropathy is a common diabetic complication and the main cause of death in patients with diabetes. Research has aimed to find an ideal drug with minimal side effects for treating this disease. Banana peel has been shown to be anti-diabetic, with lupenone isolated from banana peel exhibiting antidiabetic and anti-inflammatory activities; However, the effects of lupenone on type 2 diabetic nephropathy are largely unknown. PURPOSE: This study aimed to investigate the ameliorative effect of lupenone on type 2 diabetic nephropathy, and its mechanism from both anti-inflammatory and anti-fibrotic perspectives. METHODS: Spontaneous type 2 diabetic nephropathy db/db mouse models were given three levels of lupenone (24 or 12 or 6 mg/kg/d) via intragastric administration for six weeks, and irbesartan treatment was used for the positive control group. We explored the effects and mechanism of lupenone action using enzyme-linked immunosorbent assay, automatic biochemical analyzer, hematoxylin-eosin and Masson staining, real time-PCR, and western blotting. Concurrently, a high-sugar and high-fat diet combined with a low-dose streptozotocin-induced type 2 diabetic nephropathy rat model was used for confirmatory research. RESULTS: Lupenone administration maintained the fasting blood glucose; reduced glycosylated hemoglobin, insulin, and 24 h proteinuria levels; and markedly regulated changes in biochemical indicators associated with kidney injury in serum and urine (including 24 h proteinuria, micro-albumin, N-acetyl-ß-d-glucosaminidase, α1-micro-globulin, creatinine, urea nitrogen, uric acid, total protein, and albumin) of type 2 diabetic nephropathy mice and rats. Hematoxylin-eosin and Masson staining as well as molecular biology tests revealed that inflammation and fibrosis are the two key processes affected by lupenone treatment. Lupenone protected type 2 diabetic nephropathy kidneys by regulating the NF-κB-mediated inflammatory response and TGF-ß1/Smad/CTGF pathway-associated fibrosis. CONCLUSION: Lupenone has potential as an innovative drug for preventing and treating diabetic nephropathy. Additionally, it has great value for the utilization of banana peel resources.

PYK2 senses calcium through a disordered dimerization and calmodulin-binding element.

Multidomain kinases use many ways to integrate and process diverse stimuli. Here, we investigated the mechanism by which the protein tyrosine kinase 2-beta (PYK2) functions as a sensor and effector of cellular calcium influx. We show that the linker between the PYK2 kinase and FAT domains (KFL) encompasses an unusual calmodulin (CaM) binding element. PYK2 KFL is disordered and engages CaM through an ensemble of transient binding events. Calcium increases the association by promoting structural changes in CaM that expose auxiliary interaction opportunities. KFL also forms fuzzy dimers, and dimerization is enhanced by CaM binding. As a monomer, however, KFL associates with the PYK2 FERM-kinase fragment. Thus, we identify a mechanism whereby calcium influx can promote PYK2 self-association, and hence kinase-activating trans-autophosphorylation. Collectively, our findings describe a flexible protein module that expands the paradigms for CaM binding and self-association, and their use for controlling kinase activity.

Highly Efficient Generation of Pigs Harboring a Partial Deletion of the CD163 SRCR5 Domain, Which Are Fully Resistant to Porcine Reproductive and Respiratory Syndrome Virus 2 Infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) 1 and 2 differ in their recognition of CD163. Substitution of porcine CD163 SRCR5 domain with a human CD163-like SRCR8 confers resistance to PRRSV 1 but not PRRSV 2. The deletion of CD163 SRCR5 has been shown to confer resistance to PRRSV 1 in vivo and both PRRSV 1 and 2 in vitro. However, the anti-PRRSV 2 activity of modifying the CD163 SRCR5 domain has not yet been reported. Here, we describe the highly efficient generation of two pig breeds (Liang Guang Small Spotted and Large White pigs) lacking a short region of CD163 SRCR5, including the ligand-binding pocket. We generated a large number of gene-edited Large White pigs of the F0 generation for use in viral challenge studies. The results of this study show that these pigs are completely resistant to infection by species 2 PRRSV, JXA1, and MY strains. There were no clinical symptoms, pathological abnormalities, viremia, or anti-PRRSV antibodies in the CD163 SRCR5-edited pigs compared to wild-type controls after viral challenge. Porcine alveolar macrophages (PAMs) isolated from CD163 SRCR5-edited Large White pigs also displayed resistance to PRRSV in vitro. In addition, CD163 SRCR5-edited PAMs still exhibited a cytokine response to PRRSV infection, and no significant difference was observed in cytokine expression compared to wild-type PAMs. Taken together, these data suggest that CD163 SRCR5-edited pigs are resistant to PRRSV 2, providing a basis for the establishment of PRRSV-resistant pig lines for commercial application and further investigation of the essential region of SRCR5 involved in virus infection.

Inhibitory effect of iota-carrageenan on porcine reproductive and respiratory syndrome virus in vitro.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen and causes significant economic losses to the swine industry worldwide each year. Current vaccination strategies do not effectively prevent and control the virus. Consequently, it is necessary to develop novel antiviral strategies. Carrageenan, extracted from marine red algae, exhibits anti-coagulant, anti-tumour, anti-virus and immunomodulatory activities. METHODS: We investigate the inhibitory effect of iota-carrageenan (CG) on PRRSV strain CH-1a via antiviral assay and viral binding, entry and release assays. RESULTS: We found that CG effectively inhibited CH-1a replication at mRNA and protein levels in both Marc-145 cells and porcine alveolar macrophages (PAMs). The antiviral activity of CG occurred during viral attachment and entry in virus life cycle. In addition, CG suppressed viral release in Marc-145 cells, as well as blocked CH-1a-induced apoptosis during the late period of infection. Furthermore, CG inhibited CH-1a-induced NF-κB activation, thus interfering with cytokine production in Marc-145 cells and PAMs, which contributes to its anti-PRRSV activity. CONCLUSIONS: Taken together, our data imply that CG might be an ideal candidate that is worthwhile developing into a new anti-PRRSV prophylactic and therapeutic drug.

Chlorine dioxide inhibits the replication of porcine reproductive and respiratory syndrome virus by blocking viral attachment.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a great economic loss to the swine industry globally. Current prevention and treatment measures are not effective to control the outbreak and spread of porcine reproductive and respiratory syndrome (PRRS). In other words, new antiviral strategies are urgently needed. Chlorine dioxide (ClO2) is regarded as a broad-spectrum disinfectant with strong inhibitory effects on microbes and parasites. The purpose of this study was to evaluate the inhibitory effects and underlying molecular mechanisms of ClO2 against PRRSV infection in vitro. Here, we identified ClO2 (the purity is 99%) could inhibit the infection and replication of PRRSV in both Marc-145 cells and porcine alveolar macrophages (PAMs). ClO2 could block PRRSV binding to cells rather than internalization and release, suggesting that ClO2 blocks the first stage of the virus life cycle. We also demonstrated that the inhibition exerted by ClO2 was attributed to the degradation of PRRSV genome and proteins. Moreover, we confirmed that ClO2 could decrease the expression of inflammatory cytokines induced by PRRSV. In summary, ClO2 is an efficient agent and potently suppressed PRRSV infection in vitro.

CD163ΔSRCR5 MARC-145 Cells Resist PRRSV-2 Infection via Inhibiting Virus Uncoating, Which Requires the Interaction of CD163 With Calpain 1.

Porcine alveolar macrophages without the CD163 SRCR5 domain are resistant to porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, whether the deletion of CD163 SRCR5 in MARC-145 cells confers resistance to PRRSV and interaction of which of the host proteins with CD163 is involved in virus uncoating remain unclear. Here we deleted the SRCR5 domain of CD163 in MARC-145 cells using CRISPR/Cas9 to generate a CD163ΔSRCR5 MARC-145 cell line. The modification of CD163 had no impact on CD163 expression. CD163ΔSRCR5 cells were completely resistant to infection by PRRSV-2 strains Li11, CHR6, TJM, and VR2332. The modified cells showed no cytokine response to PRRSV-2 infection and maintained normal cell vitality comparable with the WT cells. The resistant phenotype of the cells was stably maintained through cell passages. There were no replication transcription complexes in the CD163ΔSRCR5 cells. SRCR5 deletion did not disturb the colocalization of CD163 and PRRSV-N in early endosomes (EE). However, the interaction of the viral proteins GP2a, GP3, or GP5 with CD163, which is involved in virus uncoating was affected. Furthermore, 77 CD163-binding cellular proteins affected by the SRCR5 deletion were identified by LC-MS/MS. Inhibition of calpain 1 trapped the virions in EE and forced then into late endosomes but did not block viral attachment and internalization, suggesting that calpain 1 is involved in the uncoating. Overall, CD163ΔSRCR5 MARC-145 cells are fully resistant to PRRSV-2 infection and calpain 1 is identified as a novel host protein that interacts with CD163 to facilitate PRRSV uncoating.

Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Heparan sulfate (HS) is used by PRRSV for initial attachment to target cells. However, the role of HS in the late phase of PRRSV infection and the mechanism of virus release from host cells remain largely unknown. In this study, we showed that PRRSV infection caused a decrease in HS expression and upregulated heparanase, the only known enzyme capable of degrading HS. We subsequently demonstrated that the NF-κB signaling pathway and cathepsin L protease were involved in regulation of PRRSV infection-induced heparanase. In addition, we found that ablation of heparanase expression using small interfering RNA duplexes increased cell surface expression of HS and suppressed PRRSV replication and release, whereas overexpression of heparanase reduced HS surface expression and enhanced PRRSV replication and release. These data suggest that PRRSV activates NF-κB and cathepsin L to upregulate and process heparanase, and then the active heparanase cleaves HS, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes great economic losses each year to the pig industry worldwide. The molecular mechanism of PRRSV release from host cells largely remains a mystery. In this study, we demonstrate that PRRSV activates NF-κB and cathepsin L to upregulate and process heparanase, and then the active heparanase is released to the extracellular space and exerts enzymatic activity to cleave heparan sulfate, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.

Evaluation of the characteristics of hepatic focal nodular hyperplasia: correlation between dynamic contrast-enhanced multislice computed tomography and pathological findings.

OBJECTIVE: To evaluate the characteristics of enhancement of focal nodular hyperplasia (FNH) of the liver by analyzing the dynamic contrast-enhanced multislice computed tomography (MSCT) features and correlating them with pathological findings. PATIENTS AND METHODS: Nine males and 16 females with pathologically confirmed FNH and complete preoperative contrast-enhanced MSCT data were recruited for this study. The imaging features of FNH on the pre- and postcontrast MSCT were analyzed by two experienced radiologists by consensus. RESULTS: Pathology showed central scars and abnormal blood vessels in 17 and 21 of 25 lesions, respectively, while MSCT with multiphase enhancement showed central scars in eight of the 17 lesions (47.1%) and abnormal arteries or draining veins in 13 of the 21 lesions (61.9%). Furthermore, abnormal draining veins in five lesions were found to be diagnostic, which is another important finding. CONCLUSION: Multiphase scanning can provide the panorama of FNH lesions and reveal their enhancement patterns and pathological characteristics. Abnormal blood vessels within or around the lesion are demonstrated more often than central scar, and both should be observed for FNH diagnosis.

Intrahepatic mRNA levels of type I interferon receptor and interferon-stimulated genes in genotype 1b chronic hepatitis C. Association between IFNAR1 mRNA level and sustained response to interferon therapy.

OBJECTIVE: The aim of this study was to determine the association between pretreatment intrahepatic mRNA levels of interferon receptor and interferon-stimulated genes and response to interferon therapy for genotype 1b chronic hepatitis C. METHODS: Forty-four patients with genotype 1b chronic hepatitis C who underwent liver biopsy and then received interferon therapy participated in this study. Pretreatment intrahepatic mRNA levels of interferon receptor genes (IFNAR1, IFNAR2b, and IFNAR2c) and interferon-stimulated genes (OAS1 and PKR) were quantified by competitive polymerase chain reaction. RESULTS: In the genes examined, only IFNAR1 mRNA level was significantly higher in patients with sustained virological and biochemical response to interferon therapy versus those with nonsustained response (p < 0.01). Moreover, mRNA expression ratios of IFNAR1 to IFNAR2 were also significantly higher in patients with sustained virological and biochemical response to IFN therapy (p < 0.01 and p < 0.05, respectively). On the other hand, mRNA levels of IFNAR2b, IFNAR2c, and PKR were significantly higher in patients with histologically active or advanced liver rather than patients with mild or less advanced liver. CONCLUSIONS: High intrahepatic mRNA levels of IFNAR1 and mRNA ratio of IFNAR1 to IFNAR2 before treatment may be associated with a favorable response to interferon therapy.

Long-term monitoring of platelet count, as a non-invasive marker of hepatic fibrosis progression and/or regression in patients with chronic hepatitis C after interferon therapy.

BACKGROUND: Platelet count has been shown to correlate with the hepatic fibrosis stage in chronic hepatitis C (CHC). The aim of the present study was to assess hepatic fibrosis progression or regression of CHC patients by long-term monitoring of the platelet count. METHODS: A total of 429 interferon (IFN)-treated CHC patients were studied. Follow-up data on the platelet count were collected every 6 months after IFN therapy. The IFN response was defined as follows: complete responders (CR, n = 121) demonstrating persistent clearance of serum hepatitis C virus (HCV) RNA; biochemical responders (BR, n = 94) demonstrating alanine aminotransferase (ALT) normalization for >/=6 months without eradication of HCV-RNA; and non-responder (NR, n = 214) demonstrating all other patterns. RESULTS: In comparison with the baseline level, mean platelet count increased in the CR group from 0.5 years after IFN therapy (for each point, P < 0.01), but significantly decreased in the NR group from 1 year after IFN therapy (for each point, P < 0.01). In the BR group, an increase in mean platelet count was observed from 0.5 to 3.5 years following IFN therapy (for each point, P < 0.01), followed by a gradual decrease. CONCLUSION: An increase from baseline values in platelet count was observed, regardless of the presence of HCV-RNA, in both the CR and BR groups, suggesting the importance of ALT normalization in preventing hepatic fibrosis progression in IFN-treated CHC patients.