VGLUT2 and APP family: unraveling the neurobiochemical mechanisms of neurostimulation therapy to STZ-induced diabetes and neuropathy.

Diabetic Peripheral Neuropathy (DPN) poses an escalating threat to public health, profoundly impacting well-being and quality of life. Despite its rising prevalence, the pathogenesis of DPN remains enigmatic, and existing clinical interventions fall short of achieving meaningful reversals of the condition. Notably, neurostimulation techniques have shown promising efficacy in alleviating DPN symptoms, underscoring the imperative to elucidate the neurobiochemical mechanisms underlying DPN. This study employs an integrated multi-omics approach to explore DPN and its response to neurostimulation therapy. Our investigation unveiled a distinctive pattern of vesicular glutamate transporter 2 (VGLUT2) expression in DPN, rigorously confirmed through qPCR and Western blot analyses in DPN C57 mouse model induced by intraperitoneal Streptozotocin (STZ) injection. Additionally, combining microarray and qPCR methodologies, we revealed and substantiated variations in the expression of the Amyloid Precursor Protein (APP) family in STZ-induced DPN mice. Analyzing the transcriptomic dataset generated from neurostimulation therapy for DPN, we intricately explored the differential expression patterns of VGLUT2 and APPs. Through correlation analysis, protein-protein interaction predictions, and functional enrichment analyses, we predicted the key biological processes involving VGLUT2 and the APP family in the pathogenesis of DPN and during neurostimulation therapy. This comprehensive study not only advances our understanding of the pathogenesis of DPN but also provides a theoretical foundation for innovative strategies in neurostimulation therapy for DPN. The integration of multi-omics data facilitates a holistic view of the molecular intricacies of DPN, paving the way for more targeted and effective therapeutic interventions.

Network pharmacology study to explore the multiple molecular mechanism of SH003 in the treatment of non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of human death worldwide. Herbal prescription SH003 has been developed to treat several cancers including NSCLC. Due to the multi-component nature of SH003 with multiple targets and pathways, a network pharmacology study was conducted to analyze its active compounds, potential targets, and pathways for the treatment of NSCLC. METHODS: We systematically identified oral active compounds within SH003, employing ADME criteria-based screening from TM-MC, OASIS, and TCMSP databases. Concurrently, SH003-related and NSCLC-associated targets were amalgamated from various databases. Overlapping targets were deemed anti-NSCLC entities of SH003. Protein-protein interaction networks were constructed using the STRING database, allowing the identification of pivotal proteins through node centrality measures. Empirical validation was pursued through LC-MS analysis of active compounds. Additionally, in vitro experiments, such as MTT cell viability assays and western blot analyses, were conducted to corroborate network pharmacology findings. RESULTS: We discerned 20 oral active compounds within SH003 and identified 239 core targets shared between SH003 and NSCLC-related genes. Network analyses spotlighted 79 hub genes, including TP53, JUN, AKT1, STAT3, and MAPK3, crucial in NSCLC treatment. GO and KEGG analyses underscored SH003's multifaceted anti-NSCLC effects from a genetic perspective. Experimental validations verified SH003's impact on NSCLC cell viability and the downregulation of hub genes. LC-MS analysis confirmed the presence of four active compounds, namely hispidulin, luteolin, baicalein, and chrysoeriol, among the eight compounds with a median of > 10 degrees in the herb-compounds-targets network in SH003. Previously unidentified targets like CASP9, MAPK9, and MCL1 were unveiled, supported by existing NSCLC literature, enhancing the pivotal role of empirical validation in network pharmacology. CONCLUSION: Our study pioneers the harmonization of theoretical predictions with practical validations. Empirical validation illuminates specific SH003 compounds within NSCLC, simultaneously uncovering novel targets for NSCLC treatment. This integrated strategy, accentuating empirical validation, establishes a paradigm for in-depth herbal medicine exploration. Furthermore, our network pharmacology study unveils fresh insights into SH003's multifaceted molecular mechanisms combating NSCLC. Through this approach, we delineate active compounds of SH003 and target pathways, reshaping our understanding of its therapeutic mechanisms in NSCLC treatment.

Potential mechanism of Qinggong Shoutao pill alleviating age-associated memory decline based on integration strategy.

CONTEXT: Qinggong Shoutao Wan (QGSTW) is a pill used as a traditional medicine to treat age-associated memory decline (AAMI). However, its potential mechanisms are unclear. OBJECTIVE: This study elucidates the possible mechanisms of QGSTW in treating AAMI. MATERIALS AND METHODS: Network pharmacology and molecular docking approaches were utilized to identify the potential pathway by which QGSTW alleviates AAMI. C57BL/6J mice were divided randomly into control, model, and QGSTW groups. A mouse model of AAMI was established by d-galactose, and the pathways that QGSTW acts on to ameliorate AAMI were determined by ELISA, immunofluorescence staining and Western blotting after treatment with d-gal (100 mg/kg) and QGSTW (20 mL/kg) for 12 weeks. RESULTS: Network pharmacology demonstrated that the targets of the active components were significantly enriched in the cAMP signaling pathway. AKT1, FOS, GRIN2B, and GRIN1 were the core target proteins. QGSTW treatment increased the discrimination index from -16.92 ± 7.06 to 23.88 ± 15.94% in the novel location test and from -19.54 ± 5.71 to 17.55 ± 6.73% in the novel object recognition test. ELISA showed that QGSTW could increase the levels of cAMP. Western blot analysis revealed that QGSTW could upregulate the expression of PKA, CREB, c-Fos, GluN1, GluA1, CaMKII-α, and SYN. Immunostaining revealed that the expression of SYN was decreased in the CA1 and DG. DISCUSSION AND CONCLUSIONS: This study not only provides new insights into the mechanism of QGSTW in the treatment of AAMI but also provides important information and new research ideas for the discovery of traditional Chinese medicine compounds that can treat AAMI.

Ruscogenin timing administration mitigates cerebral ischemia-reperfusion injury through regulating circadian genes and activating Nrf2 pathway.

BACKGROUND: Ruscogenin (Rus), a steroidal sapogenin extracted from Ophiopogon japonicus (L. f.) Ker-Gawl., has the effect of alleviating cerebral ischemia-reperfusion injury (IRI), acute lung injury. At present, the chronopharmacological effects of Rus are still unknown. PURPOSE: This study explored the alleviating effect and mechanism of Rus timing administration on mice cerebral IRI. METHODS: The animals in different groups were administrated Rus (10 mg/kg) by gavage at four time points (23:00-01:00, 05:00-07:00, 11:00-13:00, 17:00-19:00) respectively for 3 days. On the 4th day, middle cerebral artery occlusion (MCAO) surgery was operated during 5:00-7:00. Behavioral tests were executed and the brain was collected for infarct volume, qPCR and immunoblot detection. The levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin-1beta (IL-1ß) and inducible nitric oxide synthase (iNOS) were detected by qPCR. Glutathione (GSH), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in serum and cerebral cortex were detected. The clock genes were tested by western blot. Based on these results, 17:00-19:00 was selected to administrate Rus for further mechanism study and Nrf2 blocker group was administrated all-trans-retinoic acid (ATRA) at 14:00 for 3 days. RESULTS: Administration of Rus reduced cerebral infarcted volume, ameliorated the behavior score and upregulated the mRNA and protein expression of Per1, Bmal1, Clock, Rev-erbα, transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), quinone oxidoreductase 1 (NQO1). Administration of Rus during 17:00-19:00 had better preventive effect than other three time points. Combined administration of ATRA blunted the preventive effect of Rus. CONCLUSION: The preventive effect of Rus is affected by the time of administration, which was regulated by Nrf2 pathway. Taken together, we provide solid evidence to suggest that different administration time point affect the effectiveness of Rus in alleviating IRI.

The mechanism of Qijing Mingmu decoction on cellular senescence of conjunctivochalasis.

BACKGROUND: Qijing Mingmu decoction (QJMM), a compound Chinese medicine preparation, which consists of Lycium barbarum, Polygonatum, Ophiopogon japonicus, Poria cocos, Glycyrrhiza, Eclipta prostrata and Ligusticum striatum, has been confirmed to be effective for the treatment of conjunctivochalasis (CCH) in clinic and reduce cellular senescence. However, the underlying mechanism is still unknown. Our previous study revealed that p38-mediated cellular senescence contributed to the pathogenesis of CCH. METHODS: To explore whether p38 might be the potential therapeutic target of QJMM for CCH, CCH fibroblasts were treated with QJMM granule and then the effect of QJMM granule on the expression and promoter activity of p38α was determined by western blot and dual luciferase reporter gene assay, respectively. Meanwhile, the influence of QJMM granule on cell proliferation, oxidative stress, cellular senescence and the expression of the cellular senescence-associated genes were measured by corresponding methods. RESULTS: QJMM granule significantly decreased the protein expression of p38α and p-p38α in CCH fibroblasts in a dose-dependent manner and inhibited p38α promoter activity. QJMM granule as well as the p38 inhibitor SB203580 reduced the level of reactive oxygen species and increased the activity of superoxide dismutase in CCH fibroblasts. QJMM granule and SB203580 promoted cell proliferation and reduced the percentage of SA-ß-Gal-positive cells. The mRNA and protein expression of p53 and p21 was remarkably down-regulated by QJMM granule as well as SB203580 and that of SMP30 was up-regulated in CCH fibroblasts. CONCLUSIONS: Our findings demonstrated that QJMM granule was effective for alleviating cellular senescence of CCH fibroblasts by p38 MAPK signaling and the followed p53/p21 signaling.

Loss of Ephaptic Contacts in the Murine Thalamus during Osmotic Demyelination Syndrome.

BACKGROUND AND AIM: A murine model mimicking osmotic demyelination syndrome (ODS) revealed with histology in the relay posterolateral (VPL) and ventral posteromedial (VPM) thalamic nuclei adjoined nerve cell bodies in chronic hyponatremia, amongst the damaged 12 h and 48 h after reinstatement of osmolality. This report aims to verify and complement with ultrastructure other neurophysiology, immunohistochemistry, and molecular biochemistry data to assess the connexin-36 protein, as part of those hinted close contacts.This ODS investigation included four groups of mice: Sham (NN; n = 13), hyponatremic (HN; n = 11), those sacrificed 12 h after a fast restoration of normal natremia (ODS12h; n = 6) and mice sacrificed 48 h afterward, or ODS48 h (n = 9). Out of these, thalamic zones samples included NN (n = 2), HN (n = 2), ODS12h (n = 3) and ODS48h (n = 3). RESULTS: Ultrastructure illustrated junctions between nerve cell bodies that were immunolabeled with connexin36 (Cx36) with light microscopy and Western blots. These cell's junctions were reminiscent of low resistance junctions characterized in other regions of the CNS with electrophysiology. Contiguous neurons showed neurolemma contacts in intact and damaged tissues according to their location in the ODS zones, at 12 h and 48 h post correction along with other demyelinating alterations. Neurons and ephaptic contact measurements indicated the highest alterations, including nerve cell necrosis in the ODS epicenter and damages decreased toward the outskirts of the demyelinated zone. CONCLUSION: Ephapses contained C × 36between intact or ODS injured neurons in the thalamus appeared to be resilient beyond the core degraded tissue injuries. These could maintain intercellular ionic and metabolite exchanges between these lesser injured regions and, thus, would partake to some brain plasticity repairs.

[Preparation of mouse monoclonal antibodies against human adenovirus 55 Hexon (HAdV55 Hexon) protein].

Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.

Effects and Potential Mechanism of Zhuyu Pill Against Atherosclerosis: Network Pharmacology and Experimental Validation.

Background: Atherosclerosis (AS) is an immunoinflammatory disease associated with dyslipidemia. Zhuyu Pill (ZYP) is a classic Chinese herbal compound that has been shown to exhibit anti-inflammatory and lipid-lowering effects on AS in our previous studies. However, the underlying mechanisms by which ZYP ameliorates atherosclerosis have not yet been fully investigated. In this study, network pharmacology and in vivo experiments were conducted to explore the underlying pharmacological mechanisms of ZYP on ameliorating AS. Methods: The active ingredients of ZYP were acquired from our previous study. The putative targets of ZYP relevant to AS were obtained from TCMSP, SwissTargetPrediction, STITCH, DisGeNET, and GeneCards databases. Protein-protein interactions (PPI) network, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted using the Cytoscape software. Furthermore, in vivo experiments were carried out for target validation in apolipoprotein E (ApoE) -/- mice. Results: Animal experiments revealed that ZYP ameliorated AS mainly through lowering blood lipids, alleviating vascular inflammation, and decreasing the levels of vascular cell adhesion molecule-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), monocyte chemotactic protein-1 (MCP-1), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α). Additionally, the results of Real-Time quantitative PCR revealed that ZYP inhibited the gene expressions of mitogen-activated protein kinase (MAPK) p38, extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor kappa-B (NF-κB) p65. The Immunohistochemistry and Western blot assays showed the inhibitory effect of ZYP on the proteins level of p38, p-p38, p65, and p-p65. Conclusion: This study has provided valuable evidence on the pharmacological mechanisms of action of ZYP in ameliorating AS that will be useful for forming the rationale of future research studying the cardio-protection and anti-inflammation effects of ZYP.

Juglone Inhibits Tumor Metastasis by Regulating Stemness Characteristics and the Epithelial-to-Mesenchymal Transition in Cancer Cells both in Vitro and in Vivo.

BACKGROUND: The stemness characteristics of cancer cells, such as self-renewal and tumorigenicity, are considered to be responsible, in part, for tumor metastasis. Epithelial-to-mesenchymal transition (EMT) plays an important role in promoting both stemness and tumor metastasis. Although the traditional medicine juglone is thought to play an anticancer role by affecting cell cycle arrest, induction of apoptosis, and immune regulation, a potential function of juglone in regulating cancer cell stemness characteristics remains unknown. METHODS: In the present study, tumor sphere formation assay and limiting dilution cell transplantation assays were performed to assess the function of juglone in regulating maintenance of cancer cell stemness characteristics. EMT of cancer cells was assessed by western blot and transwell assay in vitro, and a liver metastasis model was also performed to demonstrate the effect of juglone on colorectal cancer cells in vivo. RESULTS: Data gathered indicates juglone inhibits stemness characteristics and EMT in cancer cells. Furthermore, we verified that metastasis was suppressed by juglone treatment. We also observed that these effects were, in part, achieved by inhibiting Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1). CONCLUSIONS: These results indicate that juglone inhibits maintenance of stemness characteristics and metastasis in cancer cells.

Preparation of mouse monoclonal antibodies against human adenovirus 55 Hexon (HAdV55 Hexon) protein / 细胞与分子免疫学杂志

Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.

Prostatitis No.1 traditional chinese medicine significantly exhibited anti-inflammation role on prostatitis through miR-205-5p/YES1.

Prostatitis is one common male disease with a high prevalence. Traditional Chinese medicine (TCM) has been used as an alternative method for the treatment. However, the molecular mechanism of Prostatitis No.1 Traditional Chinese Medicine (P1TCM) on prostatitis is still unclear. For this purpose, the rat models were constructed and treated with PITCM of control, model, low (10 g/kg/d), medium (20 g/kg/d), and high (40 g/kg/d), as well as the transfections of medium dosage+NC mimic, and medium dosage+miR-205-5p mimic, medium dosage+NC mimic+pc-NC, medium dosage+miR-205-5p mimic+pc-NC, and medium dosage+miR-205-5p mimic+pc-v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES1). Real-time quantitative PCR (qPCR) and western blotting analyses were carried out to evaluate the expression of miR-205-5p and YES1, respectively. The levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-alpha (TNF-α) were assessed by enzyme-linked immunosorbent assay (ELISA). The targeting role of miR-205-5p on YES1 was predicted by StarBase and verified by a dual-luciferase reporter gene assay. Results showed that the optimal treatment of P1TCM relieved the damage of prostate tissue, decreased the immunity and inflammation factors, and reduced the expression level of miR-205-5p in prostate tissue and serum. miR-205-5p mimics significantly relieved tissue damage and reduced immunity and inflammatory functions. miR-205-5p targeted YES1. YES1 was significantly upregulated in medium dosage treatment compared with Control, while downregulated compared with the Model. YES1 was also upregulated in prostatitis patients. The pc-YES1 reversed the function of the miR-205-5p mimic. In conclusion, P1TCM significantly relieved the tissue damage and reduced prostate patients' inflammatory functions through miR-205-5p/YES1, which might be essential for clinical studies.

The Human Host Defense Peptide LL-37 Overexpressed in Lung Cell Lines by Methanolic Extract of Valeriana officinalis

Abstract The present study investigated the effects of valerian methanolic extract and valerenic acid on the expression of LL-37 gene and protein in A549 and MRC5 line cells. After preparing Valerian seeds, sowing them in March 2020, and harvesting the rhizome in October 2020, the extract was prepared from the valerian rhizome by maceration method. Valerian acid content was determined using high performance liquid chromatography (HPLC). Two cell lines (A549 and MRC-5) were used to study the effects of valerian extract, and the MTT test was used to evaluate cell viability. The expression of LL-37 mRNA and protein was assessed by Real-Time PCR and western blot, respectively. In vivo safety assessments and histopathological analysis were also conducted. Data was analyzed by Graphpad Prism 8 software. Valerian methanolic extract and valerenic acid upregulated the LL-37 mRNA and protein expression in both treated cell lines. Valerenic acid showed a greater effect on upregulating LL-37 expression than valerian methanolic extract. A549 cells were more sensitive to valerian methanolic extract compared to MRC5 cells, and its cell viability was reduced. Furthermore, liver and kidney-related safety assessments showed that valerian methanolic extract had no toxic effects. In general, it was concluded that the methanolic extract of valerian as well as the resulting valerenic acid as the most important component of the extract has the ability to upregulate LL-37expression. Therefore, methanolic extract of valerian and valerenic acid can be considered for improving the immune system.

Identification of novel biomarkers and therapeutic target candidates for stasis-heat symptom pattern of acute intracerebral hemorrhage by quantitative plasma proteomics.

OBJECTIVE: To explore the novel biomarkers and therapeutic target candidates related to the stasis-heat syndrome of acute intracerebral hemorrhage (AICH). METHODS: Applying an isobaric tagging for relative and absolute quantitation-(iTRAQ-) based quantitative proteomic approach, plasma samples from AICH patients with stasis-heat, and AICH patients with non-stasis-heat and healthy control subjects were collected and analyzed to distinguish differentially expressed proteins (DEPs) correlated to AICH with stasis-heat in this block design. The standard Western blot was applied to verify DEPs. Additionally, DEPs were analyzed via bioinformatic platforms and further approved via Ingenuity Pathway Analysis (IPA). RESULTS: A total of 26 DEPs were found among AICH with the stasis-heat, AICH with non-stasis-heat, and healthy control group. The seven DEPs compared with the non-stasis-heat group are closely related to the pathogenesis of stasis heat. These proteins showed three different protein expression patterns. The alpha-1-b glycoprotein (A1BG) and copper-protein (CP) were up-regulated in the stasis-heat group, but down-regulated in the non-stasis-heat group. Compared with the non-stasis-heat group, the expression abundance of actinin, alpha 1 (ACTN1), carbonic anhydrase I (CA1), peroxiredoxin 2 (PRDX2), and vinculin (VCL) is higher in the stasis-heat group, while the CD44 is the opposite. These differences reflect that stasis-heat syndrome has more severe inflammatory immune response, coagulation disorders and damage. Bioinformatics analysis revealed that a wide variety of cellular and metabolic processes and some signaling pathways were involved in the pathophysiology of AICH with stasis-heat. AICH with stasis-heat syndrome showed more severe inflammatory reactions, tissue damage, and coagulation disorders than non-stasis heat syndrome. CONCLUSIONS: There are differences in the protein expression patterns between the stasis-heat syndrome and non-stasis-heat syndrome. These differences reflect that stasis-heat syndrome has more severe damage. CD44, CP, ACTN1, CA1, VCL, PRDX2, and A1BG could be the potential biomarkers and therapeutic target candidates of the stasis-heat subtype. This study provides a reasonable explaination for Liangxue Tongyu decoction through anti-inflammatory and brain protection treatment.

Production of Recombinant Proteins in Transgenic Tobacco Plants.

Nicotiana tabacum (the tobacco plant ) has numerous advantages for molecular farming, including rapid growth, large biomass and the possibility of both cross- and self-fertilization. In addition, genetic transformation and tissue culture protocols for regeneration of transgenic plants are well-established. Here, we describe the production of transgenic tobacco using Agrobacterium tumefaciens and the analysis of recombinant proteins, either in crude plant extracts or after purification, by enzyme-linked immunosorbent assays, sodium dodecyl sulfate polyacrylamide gel electrophoresis with western blotting and surface plasmon resonance.

Erianin suppresses proliferation and migration of cancer cells in a pyruvate carboxylase-dependent manner.

Erianin is a natural small molecule dibenzyl compound extracted from Dendrobium officinale or Dendrobium chrysotoxum. Studies show erianin has many pharmacological functions such as antioxidant, antibacterial, antiviral, improving diabetic nephropathy, relaxing bronchial smooth muscle and anti-tumor. However, the erianin-mediated molecular mechanism is elusive, and the target protein of erianin is not clear yet. Here, we screened and identified that the target protein of erianin in human hepatoma HepG2 cells is human pyruvate carboxylase, and explored the anti-tumor signal pathway regulated by erianin in several cell lines. Firstly, the interaction between human pyruvate carboxylase and erianin was studied by bioinformatics and biochemical methods. Secondly, in vitro, erianin can specifically inhibit the activity of human pyruvate carboxylase, and the purified human pyruvate carboxylase can specifically bind to the activity probe of erianin. Thirdly, human pyruvate carboxylase is highly expressed in a variety of malignant tumors, and the inhibitory effect of erianin on tumor cells is positively correlated with the expression of human pyruvate carboxylase, and erianin can selectively inhibit the activity of pyruvate carboxylase. Finally, erianin can regulate the pyruvate carboxylase-mediated Wnt/ ß- Catenin pathway. All of which provide important data for the further study of the anticancer mechanism of erianin, and lay a solid foundation for the further development and utilization of erianin.

Petasin is the main component responsible for the anti-adipogenic effect of Petasites japonicus.

Petasites japonicus is one of the most popular edible wild plants in Japan. Many biological effects of P. japonicus have been reported, including anti-allergy, anti-inflammation, and anticancer effects. Although its anti-obesity effect has been reported in several studies, the most important component responsible for this activity has not been fully elucidated. On screening the components that suppress adipocyte differentiation in 3T3-F442A cells, we found that the extract of the flower buds of P. japonicus has anti-adipogenic effect. Among the known major components of P. japonicus, petasin exhibited a potent anti-adipogenic effect at an IC50 value of 0.95 µM. Quantitative analysis revealed that the active component responsible for most of the anti-adipogenic effects of P. japonicus extract is petasin. Petasin suppressed the expression of markers of mature adipocytes (PPARγ, C/EBPα, and aP2). However, as isopetasin and petasol, analogs of petasin, did not exhibit these effects, it indicates that a double bond at the C11-C12 position and an angeloyl ester moiety were essential for the activity. Petasin affected the late stage of adipocyte differentiation and inhibited the expression of lipid synthesis factors (ACC1, FAS, and SCD1). Additionally, it was revealed that petasin could be efficiently extracted using hexane with minimal amount of pyrrolizidine alkaloids, the toxic components. These findings indicate that P. japonicus extract containing petasin could be a promising food material for the prevention of obesity.

Hyperbaric oxygen treatment improves pancreatic ß­cell function and hepatic gluconeogenesis in STZ­induced type­2 diabetes mellitus model mice.

Type­2 diabetes mellitus (T2DM) causes several complications that affect the quality of life and life span of patients. Hyperbaric oxygen therapy (HBOT) has been used to successfully treat several diseases, including carbon monoxide poisoning, ischemia, infections and diabetic foot ulcer, and increases insulin sensitivity in T2DM. The present study aimed to determine the effect of HBOT on ß­cell function and hepatic gluconeogenesis in streptozotocin (STZ)­induced type­2 diabetic mice. To establish a T2DM model, 7­week­old male C57BL/6J mice were fed a high­fat diet (HFD) and injected once daily with low­dose STZ for 3 days after 1­week HFD feeding. At the 14th week, HFD+HBOT and T2DM+HBOT groups received 1­h HBOT (2 ATA; 100% pure O2) daily from 5:00 to 6:00 p.m. for 7 days. The HFD and T2DM groups were maintained under normobaric oxygen conditions and used as controls. During HBOT, the 12­h nocturnal food intake and body weight were measured daily. Moreover, blood glucose was measured by using a tail vein prick and a glucometer. After the final HBO treatment, all mice were sacrificed to conduct molecular biology experiments. Fasting insulin levels of blood samples of sacrificed mice were measured by an ultrasensitive ELISA kit. Pancreas and liver tissues were stained with hematoxylin and eosin, while immunohistochemistry was performed to determine the effects of HBOT on insulin resistance. TUNEL was used to determine the effects of HBOT on ß­cell apoptosis, and immunoblotting was conducted to determine the ß­cell apoptosis pathway. HBOT notably reduced fasting blood glucose and improved insulin sensitivity in T2DM mice. After HBOT, ß­cell area and ß­cell mass in T2DM mice were significantly increased. HBOT significantly decreased the ß­cell apoptotic rate in T2DM mice via the pancreatic Bcl­2/caspase­3/poly(ADP­ribose) polymerase (PARP) apoptosis pathway. Moreover, HBOT improved the morphology of the liver tissue and increased hepatic glycogen storage in T2DM mice. These findings suggested that HBOT ameliorated the insulin sensitivity of T2DM mice by decreasing the ß­cell apoptotic rate via the pancreatic Bcl­2/caspase­3/PARP apoptosis pathway.

Anti-inflammatory sesquiterpenoids from the heartwood of Juniperus formosana Hayata.

Juniperus formosana Hayata (J. formosana) is a commom needlebush cultivar growing in China. Six new compounds (1-6), including four cadinene sesquiterpenoids (1-4), one abietane diterpenoid (5), and one ß-naphthol derivative (6), along with 18 known compounds (7-24) were isolated and identified through phytochemical investigation on the heartwood of J. formosana. The structures of these compounds were fully elucidated by their 1D and 2D NMR, HR-ESI-MS, UV, and IR spectral data analyses. The absolute configurations of compounds 1, 3, and 5 were confirmed by experimental and calculated electronic circular dichroism (ECD) data. Moreover, X-ray crystallographic analysis was carried out to characterize the structure of compound 4. The inhibitory effects on the nitric oxide (NO) production of all the isolated compounds were initially examined in RAW264.7 macrophages induced by lipopolysaccharide (LPS). The results showed that compounds 3 and 12 possessed significant inhibitory potency on NO generation with IC50 values of 3.41 µM and 6.15 µM among the new and known compounds, respectively. The expressions of IL-1ß, IL-6, and TNF-α were measured in LPS-stimulated RAW264.7 cells to evaluate the anti-inflammatory effects of compounds 1-24. Compounds 1-6 and 9-12 exhibited potent anti-inflammatory effects. Additionally, the expressions of p38, Erk, and IκBα proteins were further determined to explore the anti-inflammatory mechanism of the most potent compounds 3 and 12. Overall, our findings indicate the potential of J. formosana for developing medicine candidates as the treatments of inflammation.

Leonurine hydrochloride-a new drug for the treatment of menopausal syndrome: Synthesis, estrogen-like effects and pharmacokinetics.

This research aimed to investigate the estrogen-like effects of Leonurine hydrochloride (Leo). First, we developed a total synthesis of Leo from 3,4,5-trimethoxy-benzoic acid and the structure was confirmed through 1H NMR and mass spectrometry (MS). Then the estrogenic activity of Leo in vitro and in vivo was studied. The proliferation and proliferation inhibitory effects of Leo on MCF-7 cells and MDA-MB-231 cells indicate that Leo exerts estrogen-like effects through estrogen receptor α (ERα) and estrogen receptor ß((ERß) in vitro. Uterotrophic assay in juvenile mice showed that Leo has an estrogen-like effect in vivo, as it can promote the development of the uterus of juvenile mice, increase its uterine coefficient and the size of the uterine cavity, as well as the increased number of uterine glands and the thickened uterine wall. For further research, cyclophosphamide (CTX) was used to establish a mouse model of ovarian function decline. Through this model, we found that Leo can restore the estrous cycle of mice, increase the number of primordial and primary follicles in the ovaries of mice, and regulate the disordered hypothalamic-pituitary-ovarian (HPOA) axis of mice. Finally, the pharmacokinetics of Leo was studied and oral bioavailability of Leo was calculated to be 2.21%. Leo was synthesized and the estrogen-like effect in vitro and in vivo was confirmed as well as its pharmacokinetics.

Chemical structures and induction of cell death via heat shock protein inhibition of the prenylated phloroglucinol derivatives isolated from Hypericum erectum.

Four new prenylated phloroglucinol derivatives (+)-erectumol I (1a), (-)-erectumol I (1b), (-)-erectumol II (2a), and (+)-erectumol II (2b) were isolated from the methanol extracts of the whole plants of Hypericum erectum. These new compounds were isolated as a pair of enantiomers, respectively. The planar chemical structures and relative configurations of the new compounds were suggested by Cu-Kα X-ray diffraction analysis and been confirmed by high-resolution mass and 1D and 2D NMR spectroscopic data. The absolute configuration of the four new compounds were established by comparing the experimental and predicted electronic circular dichroism data. Isolated compounds 1b and 2b induced death of Adriamycin-treated HeLa cells. Their enantiomers 1a and 2a did not. In addition, the apparent mechanism of cell death of 1b was the inhibited expression of heat shock protein 105.