Exploring the Mechanism of Isoforskolin against Asthma Based on Network Pharmacology and Experimental Verification.

In this study, network pharmacology combined with biological experimental verification was utilized to screen the targets of isoforskolin (ISOF) and investigate the potential underlying mechanism of ISOF against asthma. Asthma-related targets were screened from the Genecards and DisGeNET databases. SEA and Super-PRED databases were used to obtain the targets of ISOF. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were employed to identify enriched regulatory pathways of key targets in ISOF acting on asthma. Then, a protein-protein interaction (PPI) network was constructed via STRING database and hub genes of ISOF against asthma were further screened using molecular docking. Finally, CCK-8, qPCR, and Western blotting were performed to confirm the targets of ISOF in treating asthma. A total of 96 drug potential therapeutic targets from the relevant databases were screened out. KEGG pathway enrichment analysis predicted that the target genes might be involved in the PI3K-Akt pathway. The core targets of ISOF in treating asthma were identified by the PPI network and molecular docking, including MAPK1, mTOR, and NFKB1. Consistently, in vitro experiments showed that ISOF acting on asthma was involved in inflammatory response by reducing the expression of MAPK1, mTOR, and NFKB1. The present study reveals that MAPK1, mTOR, and NFKB1 might be key targets of ISOF in asthma treatment and the anti-asthma effect might be related to the PI3K-AKT signaling pathway.

Comprehensive analysis of the m6A demethylase FTO in endothelial dysfunction by MeRIP sequencing.

N6-methyladenosine (m6A) is the most general post-transcriptional modification of eukaryotic mRNAs and long-stranded non-coding RNAs. In this process, It has been shown that FTO associates with the m6A mRNA demethylase and plays a role in diabetic vascular endothelial dysfunction. In the present study, we detected FTO protein expression in HUVECs by Western blot and found that FTO was highly expressed in all disease groups relative to the control group. To explore the mechanism of FTO in T2DM vasculopathy, we performed an analysis by methylated RNA immunoprecipitation sequencing (MeRIP-seq) to elucidate the role of aberrant m6A modification and mRNA expression in endothelial dysfunction. The results showed 202 overlapping genes with varying m6A modifications and varied mRNA expression, and GO and KEGG enrichment analysis revealed that these genes were predominantly enriched in pathways associated with T2DM complications and endothelial dysfunction. By an integrated analysis of MeRIP-seq and RNA-seq results, the IGV plots showed elevated kurtosis of downstream candidate gene modifications, which may be downstream targets for FTO to exercise biological functions. HOXA9 and PLAU mRNA expression levels were significantly down after FTO inhibition. In the current work, we set up a typological profile of the m6A genes among HUVECs as well as uncovered a hidden relationship between RNA methylation modifications for T2DM vasculopathy-associated genes. Taken together, this study indicates that endothelial functional impairment is present in T2DM patients and may be related to aberrant expression of FTO.

Identification and experimental validation of PYCARD as a crucial PANoptosis-related gene for immune response and inflammation in COPD.

Chronic inflammatory and immune responses play key roles in the development and progression of chronic obstructive pulmonary disease (COPD). PANoptosis, as a unique inflammatory cell death modality, is involved in the pathogenesis of many inflammatory diseases. We aim to identify critical PANoptosis-related biomarkers and explore their potential effects on respiratory tract diseases and immune infiltration landscapes in COPD. Total microarray data consisting of peripheral blood and lung tissue datasets associated with COPD were obtained from the GEO database. PANoptosis-associated genes in COPD were identified by intersecting differentially expressed genes (DEGs) with genes involved in pyroptosis, apoptosis, and necroptosis after normalizing and removing the batch effect. Furthermore, GO, KEGG, PPI network, WGCNA, LASSO-COX, and ROC curves analysis were conducted to screen and verify hub genes, and the correlation between PYCARD and infiltrated immune cells was analyzed. The effect of PYCARD on respiratory tract diseases and the potential small-molecule agents for the treatment of COPD were identified. PYCARD expression was verified in the lung tissue of CS/LPS-induced COPD mice. PYCARD was a critical PANoptosis-related gene in all COPD patients. PYCARD was positively related to NOD-like receptor signaling pathway and promoted immune cell infiltration. Moreover, PYCARD was significantly activated in COPD mice mainly by targeting PANoptosis. PANoptosis-related gene PYCARD is a potential biomarker for COPD diagnosis and treatment.

Oligosaccharide Blocks PAR1 (Proteinase-Activated Receptor 1)-PAR4-Mediated Platelet Activation by Binding to Thrombin Exosite II and Impairs Thrombosis.

BACKGROUND: Inappropriate activation and aggregation of platelets can lead to arterial thrombosis. Thrombin is the most potent platelet agonist that activates human platelets via two PARs (proteinase-activated receptors), PAR1 and PAR4. The aim is to study the activity and mechanism of an oligosaccharide HS-11 (the undecasaccharide, derived from sea cucumber Holothuria fuscopunctata) in inhibiting thrombin-mediated platelet activation and aggregation and to evaluate its antithrombotic activity. METHODS: Platelet activation was analyzed by detecting CD62P/P-selectin expression using flow cytometry. The HS-11-thrombin interaction and the binding site were studied by biolayer interferometry. Intracellular Ca2+ mobilization of platelets was measured by FLIPR Tetra System using Fluo-4 AM (Fluo-4 acetoxymethyl). Platelet aggregation, thrombus formation, and bleeding Assay were assessed. RESULTS: An oligosaccharide HS-11, depolymerized from fucosylated glycosaminoglycan from sea cucumber Holothuria fuscopunctata blocks the interaction of thrombin with PAR1 and PAR4 complex by directly binding to thrombin exosite II, and completely inhibits platelet signal transduction, including intracellular Ca2+ mobilization and protein phosphorylation. Furthermore, HS-11 potently inhibits thrombin-PARs-mediated platelet aggregation and reduces thrombus formation in a model of ex vivo thrombosis. CONCLUSIONS: The study firstly report that the fucosylated glycosaminoglycan oligosaccharide has antiplatelet activity by binding to thrombin exosite II, and demonstrates that thrombin exosite II plays an important role in the simultaneous activation of PAR1 and PAR4, which may be a potential antithrombotic target for effective treatment of arterial thrombosis.

STOP1 regulates CCX1-mediated Ca2+ homeostasis for plant adaptation to Ca2+ deprivation.

Calcium (Ca) is essential for plant growth and stress adaptation, yet its availability is often limited in acidic soils, posing a major threat to crop production. Understanding the intricate mechanisms orchestrating plant adaptation to Ca deficiency remains elusive. Here, we show that the Ca deficiency-enhanced nuclear accumulation of the transcription factor SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) in Arabidopsis thaliana confers tolerance to Ca deprivation, with the global transcriptional responses triggered by Ca deprivation largely impaired in the stop1 mutant. Notably, STOP1 activates the Ca deprivation-induced expression of CATION/Ca2+ EXCHANGER 1 (CCX1) by directly binding to its promoter region, which facilitates Ca2+ efflux from endoplasmic reticulum to cytosol to maintain Ca homeostasis. Consequently, the constitutive expression of CCX1 in the stop1 mutant partially rescues the Ca deficiency phenotype by increasing Ca content in the shoots. These findings uncover the pivotal role of the STOP1-CCX1 axis in plant adaptation to low Ca, offering alternative manipulating strategies to improve plant Ca nutrition in acidic soils and extending our understanding of the multifaceted role of STOP1.

Gastrodin and Gastrodigenin Improve Energy Metabolism Disorders and Mitochondrial Dysfunction to Antagonize Vascular Dementia.

Vascular dementia (VD) is the second most common dementia syndrome worldwide, and effective treatments are lacking. Gastrodia elata Blume (GEB) has been used in traditional Chinese herbal medicine for centuries to treat cognitive impairment, ischemic stroke, epilepsy, and dizziness. Gastrodin (p-hydroxymethylphenyl-b-D-glucopyranoside, Gas) and Gastrodigenin (p-hydroxybenzyl alcohol, HBA) are the main bioactive components of GEB. This study explored the effects of Gas and HBA on cognitive dysfunction in VD and their possible molecular mechanisms. The VD model was established by bilateral common carotid artery ligation (2-vessel occlusion, 2-VO) combined with an intraperitoneal injection of sodium nitroprusside solution. One week after modeling, Gas (25 and 50 mg/kg, i.g.) and HBA (25 and 50 mg/kg, i.g.) were administered orally for four weeks, and the efficacy was evaluated. A Morris water maze test and passive avoidance test were used to observe their cognitive function, and H&E staining and Nissl staining were used to observe the neuronal morphological changes; the expressions of Aß1-42 and p-tau396 were detected by immunohistochemistry, and the changes in energy metabolism in the brain tissue of VD rats were analyzed by targeted quantitative metabolomics. Finally, a Hippocampus XF analyzer measured mitochondrial respiration in H2O2-treated HT-22 cells. Our study showed that Gas and HBA attenuated learning memory dysfunction and neuronal damage and reduced the accumulation of Aß1-42, P-Tau396, and P-Tau217 proteins in the brain tissue. Furthermore, Gas and HBA improved energy metabolism disorders in rats, involving metabolic pathways such as glycolysis, tricarboxylic acid cycle, and the pentose phosphate pathway, and reducing oxidative damage-induced cellular mitochondrial dysfunction. The above results indicated that Gas and HBA may exert neuroprotective effects on VD by regulating energy metabolism and mitochondrial function.

Donepezil down-regulates propionylation, 2-hydroxyisobutyrylation, butyrylation, succinylation, and crotonylation in the brain of bilateral common carotid artery occlusion-induced vascular dementia rats.

Vascular dementia (VaD), caused by stroke or small vessel disease, is the second-most common type of dementia after Alzheimer's disease (AD). Donepezil is an acetylcholinesterase inhibitor that is currently used in patients with mild to moderate AD, and has recently been shown to improve cognitive performance in patients with VaD. In this study, we evaluated the effects of donepezil on VaD, and investigated the underlying molecular mechanisms of action. VaD was established by ligation of the bilateral common carotid artery occlusion (BCCAO). Executive function was tested by the Morris water maze (MWM) test and the attentional set shifting task (ASST). Our results showed that donepezil improved executive dysfunction and cognitive flexibility in BCCAO rats. In addition, we showed that donepezil treatment decreased the level of Aß1-42 in BCCAO rats by enzyme-linked immunosorbent assay. Post-translational modifications (PTMs) are known to be critical mechanisms in the regulation of various cellular processes. Furthermore, PTMs have been linked to the central nervous system, which highlights the importance of PTMs in neurodegenerative diseases. In this study, we used western blot analysis to identify several novel PTMs in the hippocampus of BCCAO rats that were treated with or without donepezil. The data revealed that lysine propionylation, 2-hydroxyisobutyrylation, butyrylation, succinylation, and crotonylation were elevated in the hippocampus of BCCAO rats when compared to sham rats. This increase was abolished by donepezil treatment. Taken together, we speculate that donepezil treatment improves cognitive function in our animal model of VaD, possibly by reducing aberrant acyl-PTMs.

Gastrodin Alleviates Vascular Dementia in a 2-VO-Vascular Dementia Rat Model by Altering Amyloid and Tau Levels.

Vascular dementia (VaD) is the second most common type of dementia and has become a major public health challenge as the global population ages. VaD is caused by cerebrovascular disease, and most patients with VaD have been reported to also have Alzheimer's pathologies, which is the formation of neurofibrillary tangles and amyloid plaques that are mainly composed of hyperphosphorylated Tau and amyloid ß (Aß) respectively. However, the mechanisms of VaD are not completely understood, and very few drugs are available to treat this condition. Gastrodin (Gas) is the main bioactive component of the traditional Chinese herbal plant named Tian Ma (Gastrodia elata), and it has been used to treat neurasthenia in the clinical practice of Chinese Medicine for many years. Here, we hypothesize that Gas alleviates VaD in a rat model of permanent bilateral common carotid artery occlusion (2-VO)-induced VaD. Based on the results of the Morris water maze test and attention set shift test, either 22.5 or 90 mg/kg/day Gas improved the executive dysfunction and memory impairment of 2-VO rats following an intragastric administration for 4 weeks. Both 22.5 and 90 mg/kg/day Gas reduced Aß1-40 and Aß1-42 plaques in plasma and hippocampus of 2-VO rats. Mechanistically, in 2-VO rats, treatment with Gas (90 mg/kg/day) suppressed Aß plaque deposition by decreasing the hippocampus levels of phosphorylated Tau. Thus, Gas ameliorated the cognitive deficits of 2-VO rats by inhibiting the abnormal phosphorylation of Aß and Tau.

Putrescine alleviates aluminum toxicity in rice (Oryza sativa) by reducing cell wall Al contents in an ethylene-dependent manner.

Aluminum (Al3+ ) toxicity in acidic soils limits crop productivity worldwide. In this study, we found that putrescine (PUT) significantly alleviates Al toxicity in rice roots. The addition of 0.1 mM PUT promoted root elongation and reduced the Al content in the root apices of Nipponbare (Nip) and Kasalath (Kas) rice under Al toxicity conditions. Exogenous treatment with PUT reduced the cell wall Al content by reducing polysaccharide (pectin and hemicellulose) levels and pectin methylesterase (PME) activity in roots and decreased the translocation of Al from the external environment to the cytoplasm by downregulating the expression of OsNRAT1, which responsible to encode an Al transporter protein Nrat1 (Nramp aluminum transporter 1). The addition of PUT under Al toxicity conditions significantly inhibited ethylene emissions and suppressed the expression of genes involved in ethylene biosynthesis. Treatment with the ethylene precursor 1-aminocylopropane-1-carboxylic acid (ACC) significantly improved ethylene emission, inhibited root elongation, increased the Al accumulation in root tips and the root cell wall, and increased cell wall pectin and hemicellulose contents in both rice cultivars under Al toxicity conditions. The ethylene biosynthesis antagonist aminoethoxyvinylglycine (AVG, inhibitor of the ACC synthase) had the opposite effect and reduced PME activity. Together, our results show that PUT decreases the cell wall Al contents by suppressing ethylene emissions and decreases the symplastic Al levels by downregulating OsNRAT1 in rice.

Plasma contact activation by a fucosylated chondroitin sulfate and its structure-activity relationship study.

Plasma contact system is the initial part of both the intrinsic coagulation pathway and kallikrein-kinin pathway, which mainly involves three proteins: coagulation factor XII (FXII), prekallikrein (PK) and high-molecular weight kininogen. Fucosylated chondroitin sulfate (FCS) is a unique sulfated glycosaminoglycan (GAG) composed of a chondroitin sulfate-like backbone and sulfated fucose branches. The native FCS was preliminary found to cause undesired activation of the plasma contact system. How this unusual GAG functions in this process remains to be clarified. Herein, the relationship between its structure, plasma contact activation and its effects on the PK-FXII reciprocal activation loop were studied. The recalcification time assay indicated that the FCS at high concentration could be procoagulant which may be attributed to its contact activation activity. The structure-activity relationship study indicated that its high molecular weight and distinct fucose side chains are required for contact activation by FCS, although the sulfate substitution types of its side chains have less impact. In human plasma, the native FCSs potently induced FXII-dependent contact activation. However, in purified systems FCS did not significantly activate FXII per se or induce its autoactivation, whereas FCS significantly promoted the activation of PK by factor XIIa. Polysaccharide-protein interaction assays showed that FCS bound to PK with higher affinity than other contact system proteins. These data suggested that potent contact activation by FCS requires the positive feedback loop between PK and FXII. These findings contribute to better understanding of contact activation by complex GAG.

Discovery of an intrinsic tenase complex inhibitor: Pure nonasaccharide from fucosylated glycosaminoglycan.

Selective inhibition of the intrinsic coagulation pathway is a promising strategy for developing safer anticoagulants that do not cause serious bleeding. Intrinsic tenase, the final and rate-limiting enzyme complex in the intrinsic coagulation pathway, is an attractive but less explored target for anticoagulants due to the lack of a pure selective inhibitor. Fucosylated glycosaminoglycan (FG), which has a distinct but complicated and ill-defined structure, is a potent natural anticoagulant with nonselective and adverse activities. Herein we present a range of oligosaccharides prepared via the deacetylation-deaminative cleavage of FG. Analysis of these purified oligosaccharides reveals the precise structure of FG. Among these fragments, nonasaccharide is the minimum fragment that retains the potent selective inhibition of the intrinsic tenase while avoiding the adverse effects of native FG. In vivo, the nonasaccharide shows 97% inhibition of venous thrombus at a dose of 10 mg/kg in rats and has no obvious bleeding risk. This nonasaccharide may therefore serve as a novel promising anticoagulant.

Structural analysis and anticoagulant activities of the novel sulfated fucan possessing a regular well-defined repeating unit from sea cucumber.

Sulfated fucans, the complex polysaccharides, exhibit various biological activities. Herein, we purified two fucans from the sea cucumbers Holothuria edulis and Ludwigothurea grisea. Their structures were verified by means of HPGPC, FT-IR, GC-MS and NMR. As a result, a novel structural motif for this type of polymers is reported. The fucans have a unique structure composed of a central core of regular (1→2) and (1→3)-linked tetrasaccharide repeating units. Approximately 50% of the units from L. grisea (100% for H. edulis fucan) contain sides of oligosaccharides formed by nonsulfated fucose units linked to the O-4 position of the central core. Anticoagulant activity assays indicate that the sea cucumber fucans strongly inhibit human blood clotting through the intrinsic pathways of the coagulation cascade. Moreover, the mechanism of anticoagulant action of the fucans is selective inhibition of thrombin activity by heparin cofactor II. The distinctive tetrasaccharide repeating units contribute to the anticoagulant action. Additionally, unlike the fucans from marine alga, although the sea cucumber fucans have great molecular weights and affluent sulfates, they do not induce platelet aggregation. Overall, our results may be helpful in understanding the structure-function relationships of the well-defined polysaccharides from invertebrate as new types of safer anticoagulants.

Anti-HIV-1 activity and structure-activity-relationship study of a fucosylated glycosaminoglycan from an echinoderm by targeting the conserved CD4 induced epitope.

BACKGROUND: Fucosylated glycosaminoglycan (FG) is a novel glycosaminoglycan with a chondroitin sulfate-like backbone and fucose sulfate branches. The aim of this study is to investigate the mechanism and structure-activity relationships (SAR) of FG for combating HIV-1 infection. METHODS: Anti-HIV activities of FGs were assessed by a cytopathic effect assay and an HIV-1 p24 detection assay. The biomolecule interactions were explored via biolayer interferometry technology. The SAR was established by comparing its anti-HIV-1 activities, conserved CD4 induced (CD4i) epitope-dependent interactions and anticoagulant activities. RESULTS: FG efficiently and selectively inhibited the X4- and R5X4-tropic HIV-1 infections in C8166 cells with little cytotoxicity against C8166 cells and PBMCs. Our data indicated that FG bound to gp120 with nanomolar affinity and may interact with CD4i of gp120. Additionally, the CD4i binding affinity of FG was higher than that of dextran sulfate. SAR studies suggested that the unique sulfated fucose branches account for the anti-HIV-1 activity. The molecular size and present carboxyl groups of FG may also play important roles in various activities. Notably, several FG derivatives showed higher anti-HIV-1 activities and much lower anticoagulant activities than those of heparin. CONCLUSIONS: FG exhibits strong activity against X4- and R5X4-tropic HIV-1 infections. The mechanism may be related to targeting CD4i of gp120, which results in inhibition of HIV-1 entry. The carboxyl group substituted derivatives of FG (8.5-12.8kDa), might display high anti-HIV-1 activity and low anticoagulant activity. GENERAL SIGNIFICANCE: Our data supports further the investigation of FG derivatives as novel HIV-1 entry inhibitors targeting CD4i.

[Protective effects of Qizhen Jiangtang granules in diabetic nephropathy rats].

OBJECTIVE: To study the curative and protective effects of Qizhen Jiangtang Granules in the diabetic nephropathy (DN) model rats. METHODS: Healthy SD rats were fed a high-sucrose and high-fat diet and intraperitoneal injection of streptozotocin (STZ, 30 mg/kg) to establish the DN model. The rats were divided into six groups including normal control group,model group, positive control group, high-dosage group(200 mg/kg), medium-dosage group (100 mg/kg), and low-dosage group(50 mg/kg). After oral administration of Qizhen Jiangtang Granules for eight weeks, FBG,TG,TC, LDL-c, HDL-c, SCr and BUN levels in rats serum were determined, while the pathological damage of kidney tissue with PAS and HE staining were observed under microscope. RESULTS: After treatment, TG, TC, LDL-c,SCr and BUN levels were significantly decreased(P <0. 05), and HDL-c level was significantly increased(P <0. 05). The treatment also alleviated the pathological damage of kidney tissue. CONCLUSION: Qizhen Jiangtang Granules have a protective effect against kidney damage in DN model rats. The mechanism may be related to the regulation of lipid and sugar levels in serum.

[Therapeutic effect analysis of non-invasive ear moldings on correcting congenital auricular deformity in 435 ears].

Objective:To evaluate the therapeutic effect of non-invasive ear moldings on correcting congenital auricular deformity of infants. Methods:A total of 435 ears with congenital auricular deformity were treated in the department of Otorhinolaryngology of Chengdu Women and Children's Central Hospital from December 2019 to July 2023. The infants were divided into 3 groups according to the initial treatment age, i. e. , 0-30 days（group A, n=106, 175 ears）, 31-90 days（group B, n=124, 202 ears） and ≥91 days（group C, n=37, 58 ears）. All infants were corrected with Chinese ear moldings. The efficacy, complication rate and treatment duration were compared among the three groups, and the treatments of different types of auricle deformity was analyzed. Results:Through comparison of three groups, the results showed that the markedly effective and cured rate of group A was the highest, and group C was the lowest. The difference was statistically significant（P=0.008）. Specially, there was a significant decrease of after 5 months in Group C. There was no significant difference in the complication rate among the three groups（P=0.232）, and the occurrence of complications has no significant impact on treatment. Group C has the longest treatment duration and group A has the shortest. The difference was statistically significant（P<0.001）. Conclusion:Congenital auricle deformity should be early detected and intervened. The younger the age of children, the better efficacy and shorter treatment duration can be acquired. Children under 5 months still will receive a good treatment. Nevertheless, for children older than 5 months, corrective treatment may also be considered. The efficacy for malformations was significantly lower than that of deformations. The number of cases, efficacy and complication rate for different types of deformations were significantly different.

Structure and pharmacokinetics/pharmacodynamics of the anticoagulant tetradecasaccharide oHG-14 as an intrinsic tenase inhibitor.

The intrinsic tenase complex (iXase) is an attractive antithrombotic target to treat or prevent pathological thrombosis with negligible bleeding risk. Fucosylated glycosaminoglycan (FG) is a promising anticoagulant by inhibiting iXase. A depolymerized FG (dHG-5) as an anticoagulant has been approved for clinical trials. Given that dHG-5 is a multi-component drug candidate consisting of a homologous series of oligosaccharides, it is difficult to predict a clear pharmacokinetics. Here, as a major oligosaccharide component, the tetradecasaccharide (oHG-14) was purified from dHG-5 and its structure was defined as L-Fuc3S4S-α(1,3)-L-Δ4,5GlcA-α(1,3)-{D-GalNAc4S6S-ß(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-ß(1,3)-}3-D-GalNAc4S6S-ß(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-ol. oHG-14 showed potent iXase inhibitory activity in vitro and antithrombotic effect in vivo comparable to dHG-5. After single subcutaneous administration of oHG-14 at 8, 14.4 and 32.4 mg/kg to rats, the absolute bioavailability was 71.6 %-80.9 % determined by the validated bioanalytical methods. The maximum concentration (Cmax) was 3.73, 8.07, and 11.95 µg/mL, respectively, and the time reaching Cmax (Tmax) was about 1 h. oHG-14 was mainly excreted by kidney as the parent compound with the elimination kinetics of first-order linear model. Anticoagulant activity of oHG-14 was positively correlated with its concentration in rat plasma. The pharmacokinetics/pharmacodynamics (PK/PD) of oHG-14 is similar to that of dHG-5. This study could provide supportive data for the clinical trial of dHG-5 and further development of pure oligosaccharide as an antithrombotic drug candidate.

Luteolin Alleviates Diabetic Nephropathy Fibrosis Involving AMPK/NLRP3/TGF-ß Pathway.

Purpose: Luteolin is a promising candidate for diabetic nephropathy due to its potential anti-inflammatory and anti-fibrotic properties. This study explored the molecular mechanisms through which luteolin combats fibrosis in DN. Methods: Potential targets affected by luteolin and genes associated with DN were collected from databases. Overlapping targets between luteolin and diabetic nephropathy were identified through Venn analysis. A protein-protein interaction network was constructed using these common targets, and critical pathways and targets were elucidated through GO and KEGG analysis. These pathways and targets were confirmed using a streptozotocin-induced mouse model. Luteolin was administered at 45 mg/kg and 90 mg/kg. Various parameters were evaluated, including body weight, blood glucose levels, and histopathological examinations. Protein levels related to energy metabolism, inflammation, and fibrosis were quantified. Results: Fifty-three targets associated with luteolin and 36 genes related to diabetic nephropathy were extracted. The AGE-RAGE signaling pathway was the key pathway impacted by luteolin in diabetic nephropathy. Key molecular targets include TGF-ß, IL-1ß, and PPARG. Luteolin reduced body weight and blood glucose levels, lowered the left kidney index, and improved insulin and glucose tolerance. Furthermore, luteolin mitigated inflammatory cell infiltration, basement membrane thickening, and collagen deposition in the kidney. Luteolin up-regulated the protein expression of p-AMPKα (Th172) while simultaneously down-regulated the protein expression of p-NF-ĸB (p65), NLRP3, TGF-ß1, α-SMA, and Collagen I. Conclusion: Luteolin mitigated renal fibrosis by alleviating energy metabolism disruptions and inflammation by modulating the AMPK/NLRP3/TGF-ß signaling pathway.

Production and characterization of highly purified recombinant thymosin beta 4 in Escherichia coli.

Thymosin ß4 (Tß4) is a small peptide composed of 43 amino acids. It has many important biological functions, such as promoting cardiac repair and wound healing, and therefore has great potential in clinical applications. In this report, we describe a novel and efficient way to produce highly purified and active Tß4. It was expressed in a soluble form using a DsbA and hexahistindine tag in Escherichia coli (E. coli). Using high cell density cultivation, the final biomass concentration was about 50 g L(-1) dry cell weight with the expression level of the fusion protein being 40%. To obtain highly purified protein, a purification process involving a five-step column procedure was implemented. The purity of Tß4 was above 98% and all the host cell related impurities, such as endotoxin, host cell protein and residual DNA levels, were within the permissible range listed in the Chinese Pharmacopoeia. The E-rosette test demonstrated that the bioactivity of purified Tß4 was consistent with other published work. This is the first report producing highly purified Tß4 from genetically engineered sources.

Comparison of physicochemical characteristics and anticoagulant activities of polysaccharides from three sea cucumbers.

In order to search for sulfated polysaccharides in different invertebrate connective tissues and to examine their biological activities, we have isolated three types of polysaccharides from the body wall of the three sea cucumbers Holothuria edulis, Apostichopus japonicas and Holothuria nobilis. The physicochemical properties and anticoagulant activities of these polysaccharides were examined and compared. The chemical composition analysis and nuclear magnetic resonance (NMR) analysis indicate that two types of polysaccharides, sulfated fucan and fucosylated chondroitin sulfate (FuCS), were found in all of the three species and in addition a neutral glycan was observed in H. edulis. The neutral α-glucan was firstly obtained from sea cucumber. The same type of polysaccharides from different species of sea cucumbers have similar physicochemical properties and anticoagulant activities, but those of different types of glycans are significantly different, possibly due to their different monosaccharide compositions, electric charges and average molecular weights. The FuCSs have stronger anticoagulant activities than the sulfated fucans, although the molecular sizes of the FuCSs are lower than those of the sulfated fucans, whereas the neutral glucan has no activity, as expected from the absence of sulfate. Thus, anticoagulant activities of the different type of polysaccharides are likely to relate to monosaccharide composition and sulfate content. Preliminary analysis suggests that the sulfation patterns of the FuCSs may result in the difference in anticoagulant activities. Our data could help elucidate the structure-activity relationship of the sea cucumber polysaccharides.

[CpG methyltransferase induced down-regulation of claudin-7, -8 and its effects on proliferation and apoptosis of human colorectal cancer HT-29 cells].

OBJECTIVE: To explore the regulatory effect of CpG methyltransferase (M.SssI) on expression of claudin-7 and claudin-8, promoting apoptosis and inhibiting proliferation of human colorectal cancer HT-29 cells. METHODS: HT-29 cells were treated with M.SssI (50 U/ml) for 24 hours. The methylation status of claudin-7 and claudin-8 gene promoters was assayed by bisulfite sequencing PCR (BSP). Real-time PCR with SYBR green I technique was used to detect the relative expression of claudin-7 and -8 mRNA, and claudin-7 and claudin-8 proteins were tested by cell immunofluorescence and Western blotting, while the effect on cell apoptosis was assessed by Hoechst 33342 fluorescence and flow cytometry. Inhibition of cell proliferation was measured by MTT assay. RESULTS: The amounts of methylated claudin-7 and claudin-8 gene CpGs were 25, 10 in the M.SssI group, 9 and 5 in the PBS group, 0 and 3 in the 5-azacytidine group, respectively. Compared with the PBS group, Claudin-7 and -8 were significantly reduced by M.SssI (P < 0.05), but increased by 5-azacytidine (P < 0.05) at both mRNA and protein levels. Hoechst 33342 staining revealed that HT-29 cells treated with PBS and 5-azacytidine were not significantly different, showing even blue fluorescence, round shape and same cell volume. But the M.SssI group presented more apoptotic cells with intensive white fluorescence intensity. Cytometry indicated that early apoptotic index of the M.SssI group was increased by 84.7%, compared with that of the PBS group (P = 0.002). Measurement of MTT optical density demonstrated that cell growth of the M.SssI group was significantly lower than that of the PBS group (P = 0.002), with an inhibition rate of 32.1%, whereas the proliferation of 5-azacytidine group was similar to that of the PBS group (P = 0.084). CONCLUSIONS: Our findings suggest that M.SssI can down-regulate claudin-7, -8 mRNA and proteins in the human colon cancer HT-29 cells by up-regulating methylation status of claudin-7 and -8 gene promoters, and finally induce apoptosis and inhibit proliferation of the tumor cells.