Pimpinellin ameliorates macrophage inflammation by promoting RNF146-mediated PARP1 ubiquitination.

Macrophage inflammation plays a central role during the development and progression of sepsis, while the regulation of macrophages by parthanatos has been recently identified as a novel strategy for anti-inflammatory therapies. This study was designed to investigate the therapeutic potential and mechanism of pimpinellin against LPS-induced sepsis. PARP1 and PAR activation were detected by western blot or immunohistochemistry. Cell death was assessed by flow cytometry and western blot. Cell metabolism was measured with a Seahorse XFe24 extracellular flux analyzer. C57, PARP1 knockout, and PARP1 conditional knock-in mice were used in a model of sepsis caused by LPS to assess the effect of pimpinellin. Here, we found that pimpinellin can specifically inhibit LPS-induced macrophage PARP1 and PAR activation. In vitro studies showed that pimpinellin could inhibit the expression of inflammatory cytokines and signal pathway activation in macrophages by inhibiting overexpression of PARP1. In addition, pimpinellin increased the survival rate of LPS-treated mice, thereby preventing LPS-induced sepsis. Further research confirmed that LPS-induced sepsis in PARP1 overexpressing mice was attenuated by pimpinellin, and PARP1 knockdown abolished the protective effect of pimpinellin against LPS-induced sepsis. Further study found that pimpinellin can promote ubiquitin-mediated degradation of PARP1 through RNF146. This is the first study to demonstrate that pimpinellin inhibits excessive inflammatory responses by promoting the ubiquitin-mediated degradation of PARP1.

Genome-wide analysis of the U-box E3 ligases gene family in potato (Solanum tuberosum L.) and overexpress StPUB25 enhance drought tolerance in transgenic Arabidopsis.

BACKGROUND: Plant U-box (PUB) E3 ubiquitin ligases have vital effects on various biological processes. Therefore, a comprehensive and systematic identification of the members of the U-box gene family in potato will help to understand the evolution and function of U-box E3 ubiquitin ligases in plants. RESULTS: This work identified altogether 74 PUBs in the potato (StPUBs) and examined their gene structures, chromosomal distributions, and conserved motifs. There were seventy-four StPUB genes on ten chromosomes with diverse densities. As revealed by phylogenetic analysis on PUBs within potato, Arabidopsis, tomato (Solanum lycopersicum), cabbage (Brassica oleracea), rice (Oryza sativa), and corn (Zea mays), were clustered into eight subclasses (C1-C8). According to synteny analysis, there were 40 orthologous StPUB genes to Arabidopsis, 58 to tomato, 28 to cabbage, 7 to rice, and 8 to corn. In addition, RNA-seq data downloaded from PGSC were utilized to reveal StPUBs' abiotic stress responses and tissue-specific expression in the doubled-monoploid potato (DM). Inaddition, we performed RNA-seq on the 'Atlantic' (drought-sensitive cultivar, DS) and the 'Qingshu NO.9' (drought-tolerant cultivar, DT) in early flowering, full-blooming, along with flower-falling stages to detect genes that might be involved in response to drought stress. Finally, quantitative real-time PCR (qPCR) was carried out to analyze three candidate genes for their expression levels within 100 mM NaCl- and 10% PEG 6000 (w/v)-treated potato plantlets for a 24-h period. Furthermore, we analyzed the drought tolerance of StPUB25 transgenic plants and found that overexpression of StPUB25 significantly increased peroxidase (POD) activity, reduced ROS (reactive oxygen species) and MDA (malondialdehyde) accumulation compared with wild-type (WT) plants, and enhancing drought tolerance of the transgenic plants. CONCLUSION: In this study, three candidate genes related to drought tolerance in potato were excavated, and the function of StPUB25 under drought stress was verified. These results should provide valuable information to understand the potato StPUB gene family and investigate the molecular mechanisms of StPUBs regulating potato drought tolerance.

Recent advances in targeted protein degraders as potential therapeutic agents.

Targeted protein degradation (TPD) technology has gradually become widespread in the past 20 years, which greatly boosts the development of disease treatment. Contrary to small inhibitors that act on protein kinases, transcription factors, ion channels, and other targets they can bind to, targeted protein degraders could target "undruggable targets" and overcome drug resistance through ubiquitin-proteasome pathway (UPP) and lysosome pathway. Nowadays, some bivalent degraders such as proteolysis-targeting chimeras (PROTACs) have aroused great interest in drug discovery, and some of them have successfully advanced into clinical trials. In this review, to better understand the mechanism of degraders, we elucidate the targeted protein degraders according to their action process, relying on the ubiquitin-proteasome system or lysosome pathway. Then, we briefly summarize the study of PROTACs employing different E3 ligases. Subsequently, the effect of protein of interest (POI) ligands, linker, and E3 ligands on PROTAC degradation activity is also discussed in detail. Other novel technologies based on UPP and lysosome pathway have been discussed in this paper such as in-cell click-formed proteolysis-targeting chimeras (CLIPTACs), molecular glues, Antibody-PROTACs (Ab-PROTACs), autophagy-targeting chimeras, and lysosome-targeting chimeras. Based on the introduction of these degradation technologies, we can clearly understand the action process and degradation mechanism of these approaches. From this perspective, it will be convenient to obtain the development status of these drugs, choose appropriate degradation methods to achieve better disease treatment and provide basis for future research and simultaneously distinguish the direction of future research efforts.

Vitamin D Enhances Immune Effector Pathways of NK Cells Thus Providing a Mechanistic Explanation for the Increased Effectiveness of Therapeutic Monoclonal Antibodies.

Patients with diffuse large cell lymphoma who have an adequate vitamin D supply derive significantly more benefit from immuno-chemotherapy with rituximab than patients with vitamin D deficiency; this is especially true for female patients. We have already been able to show that vitamin D increases the antibody-dependent cytotoxicity (ADCC) of NK cells in a sex-dependent manner, but it is unclear how vitamin D makes NK cells more efficient. METHODS: Healthy individuals with vitamin D deficiency were supplemented with vitamin D to sufficient levels. NK cells were isolated from blood samples before and after vitamin D saturation. For transcriptome analysis, we used the Affymetrix Gene-Chip 2.0™. Gene expression analysis as well as supervised and unsupervised pathway analysis were performed. RESULTS: Among others the "NK cell-associated cytotoxicity pathway" increased after vitamin D substitution. Five IFN-α subtypes (2, 4, 6, 7 and 10) and IFN-κ were more highly expressed and are mainly responsible in these pathways. In contrast, the pathway "interferon-gamma response", as well as other sets in cytokine production and chemotaxis showed a reduction. Toll-like receptor genes (TLR-8, TLR-7, TLR-2) were downregulated and, therefore, are responsible for the decline of these pathways. The same could be shown for the "ubiquitin-ligase" pathway. CONCLUSIONS: Increased expression of several IFN-α subtypes may explain the increased ADCC of NK cells in vitamin D-replenished and otherwise healthy subjects. Other regulators of interferon production and ADCC are compensatory upregulated in compensation, such as Toll-like receptors and those of the ubiquitin ligase, and normalize after vitamin D substitution.

Metabolomics and Proteomics Responses of Largemouth Bass (Micropterus salmoides) Muscle under Organic Selenium Temporary Rearing.

Organic selenium has been widely studied as a nutritional supplement for animal feed. However, there are few studies on the effect of organic selenium on flesh quality. In this study, the effects of organic selenium (yeast selenium (YS), Se 0.002 mg/L) on the metabolism and protein expression in Micropterus salmoides muscle under temporary fasting condition (6 weeks) were investigated. The muscle structure was observed through a microscope, and regulatory pathways were analyzed using proteomics and metabolomics methods. Electron microscopy showed that YS made the muscle fibers of M. salmoides more closely aligned. Differential analysis identified 523 lipid molecules and 268 proteins. The numbers of upregulated and downregulated proteins were 178 and 90, respectively, including metabolism (46.15%), cytoskeleton (11.24%) and immune oxidative stress (9.47%), etc. Integrated analyses revealed that YS enhanced muscle glycolysis, the tricarboxylic acid cycle and oxidative phosphorylation metabolism. In the YS group, the content of eicosapentaenoic acid was increased, and that of docosahexaenoic acid was decreased. YS slowed down protein degradation by downregulating ubiquitin and ubiquitin ligase expression. These results suggest that organic selenium can improve M. salmoides muscle quality through the aforementioned pathways, which provides potential insights into the improvement of the quality of aquatic products, especially fish.

Dietary zinc inadequacy affects neurotrophic factors and proteostasis in the rat brain.

Zinc (Zn) deficiency has many adverse effects, including growth retardation, loss of appetite, vascular diseases, cognitive and memory impairment, and neurodegenerative diseases. In the current study, we investigated the hypothesis that dietary Zn inadequacy affects neurotrophic factors and proteostasis in the brain. Three-week-old Wistar/Kyoto male rats were fed either a Zn-deficient diet (D; < 1 mg Zn/kg diet; n = 18) or pair-fed with the control diet (C; 48 mg Zn/kg diet; n = 9) for 4 weeks. Subsequently, the rats in the D group were subdivided into two groups (n = 9), in which one group continued to receive a Zn-deficient diet, whereas the other received a Zn-supplemented diet (R; 48 mg Zn/kg diet) for 3 more weeks, after which the rats were sacrificed to collect their brain tissue. Markers of endoplasmic reticulum stress, ubiquitin-proteasome system, autophagy, and apoptosis, along with neurotrophic factors, were investigated by immunoblotting. Proteasomal activity was analyzed by the spectrofluorometric method. The results showed an altered ubiquitin-proteasome system and autophagy components and increased gliosis, endoplasmic reticulum stress, and apoptosis markers in Zn-deficient rats compared with the control group. Zinc repletion for 3 weeks could partially restore these alterations, indicating a necessity for an extended duration of Zn supplementation. In conclusion, a decline in Zn concentrations below a critical threshold may trigger multiple pathways, leading to brain-cell apoptosis.

Isorhamnetin and anti-PD-L1 antibody dual-functional mesoporous silica nanoparticles improve tumor immune microenvironment and inhibit YY1-mediated tumor progression.

BACKGROUND: The immune checkpoint inhibitor (ICI) anti-PD-L1 monoclonal antibody can inhibit the progress of hepatocellular carcinoma (HCC). Epithelial-mesenchymal transformation (EMT) can promote tumor migration and the formation of immune-suppression microenvironment, which affects the therapeutic effect of ICI. Yin-yang-1 (YY1) is an important transcription factor regulating proliferation, migration and EMT of tumor cells. This work proposed a drug-development strategy that combined the regulation of YY1-mediated tumor progression with ICIs for the treatment of HCC. METHODS: We first studied the proteins that regulated YY1 expression by using pull-down, co-immunoprecipitation, and duo-link assay. The active compound regulating YY1 content was screened by virtual screening and cell-function assay. Isorhamnetin (ISO) and anti-PD-L1 antibody dual-functional mesoporous silica nanoparticles (HMSN-ISO@ProA-PD-L1 Ab) were prepared as an antitumor drug to play a synergistic anti-tumor role. RESULTS: YY1 can specifically bind with the deubiquitination enzyme USP7. USP7 can prevent YY1 from ubiquitin-dependent degradation and stabilize YY1 expression, which can promote the proliferation, migration and EMT of HCC cells. Isorhamnetin (ISO) were screened out, which can target USP7 and promote YY1 ubiquitin-dependent degradation. The cell experiments revealed that the HMSN-ISO@ProA-PD-L1 Ab nanoparticles can specifically target tumor cells and play a role in the controlled release of ISO. HMSN-ISO@ProA-PD-L1 Ab nanoparticles inhibited the growth of Hepa1-6 transplanted tumors and the effect was better than that of PD-L1 Ab treatment group and ISO treatment group. HMSN-ISO@ProA-PD-L1 Ab nanoparticles also exerted a promising effect on reducing MDSC content in the tumor microenvironment and promoting T-cell infiltration in tumors. CONCLUSIONS: The isorhamnetin and anti-PD-L1 antibody dual-functional nanoparticles can improve tumor immune microenvironment and inhibit YY1-mediated tumor progression. This study demonstrated the possibility of HCC treatment strategies based on inhibiting USP7-mediated YY1 deubiquitination combined with anti-PD-L1 monoclonal Ab.

Morusinol extracted from Morus alba induces cell cycle arrest and apoptosis via inhibition of DNA damage response in melanoma by CHK1 degradation through the ubiquitin-proteasome pathway.

BACKGROUD: Flavonoids have a variety of biological activities, such as anti-inflammation, anti-tumor, anti-thrombosis and so on. Morusinol, as a novel isoprene flavonoid extracted from Morus alba root barks, has the effects of anti-arterial thrombosis and anti-inflammatory in previous studies. However, the anti-cancer mechanism of morusinol remains unclear. PURPOSE: In present study, we mainly studied the anti-tumor effect of morusinol and its mode of action in melanoma. METHODS: The anti-cancer effect of morusinol on melanoma were evaluated by using the MTT, EdU, plate clone formation and soft agar assay. Flow cytometry was used for detecting cell cycle and apoptosis. The É£-H2AX immunofluorescence and the alkaline comet assay were used to detect DNA damage and the Western blotting analysis was used to investigate the expressions of DNA-damage related proteins. Ubiquitination and turnover of CHK1 were also detected by using the immunoprecipitation assay. The cell line-derived xenograft (CDX) mouse models were used in vivo to evaluate the effect of morusinol on tumorigenicity. RESULTS: We demonstrated that morusinol not only had the ability to inhibit cell proliferation, but also induced cell cycle arrest at G0/G1 phase, caspase-dependent apoptosis and DNA damage in human melanoma cells. In addition, morusinol effectively inhibited the growth of melanoma xenografts in vivo. More strikingly, CHK1, which played an important role in maintaining the integrity of cell cycle, genomic stability and cell viability, was down-regulated in a dose- and time-dependent manner after morusinol treatment. Further research showed that CHK1 was degraded by the ubiquitin-proteasome pathway. Whereafter, morusinol-induced cell cycle arrest, apoptosis and DNA damage were partially salvaged by overexpressing CHK1 in melanoma cell lines. Herein, further experiments demonstrated that morusinol increased the sensitivity of dacarbazine (DTIC) to chemotherapy for melanoma in vitro and in vivo. CONCLUSION: Morusinol induces CHK1 degradation through the ubiquitin-proteasome pathway, thereby inducing cell cycle arrest, apoptosis and DNA damage response in melanoma. Our study firstly provided a theoretical basis for morusinol to be a candidate drug for clinical treatment of cancer, such as melanoma, alone or combinated with dacarbazine.

Emergence of consciousness from anesthesia through ubiquitin degradation of KCC2 in the ventral posteromedial nucleus of the thalamus.

The emergence of consciousness from anesthesia, once assumed to be a passive process, is now considered as an active and controllable process. In the present study, we show in mice that, when the brain is forced into a minimum responsive state by diverse anesthetics, a rapid downregulation of K+/Cl- cotransporter 2 (KCC2) in the ventral posteromedial nucleus (VPM) serves as a common mechanism by which the brain regains consciousness. Ubiquitin-proteasomal degradation is responsible for KCC2 downregulation, which is driven by ubiquitin ligase Fbxl4. Phosphorylation of KCC2 at Thr1007 promotes interaction between KCC2 and Fbxl4. KCC2 downregulation leads to γ-aminobutyric acid type A receptor-mediated disinhibition, enabling accelerated recovery of VPM neuron excitability and emergence of consciousness from anesthetic inhibition. This pathway to recovery is an active process and occurs independent of anesthetic choice. The present study demonstrates that ubiquitin degradation of KCC2 in the VPM is an important intermediate step en route to emergence of consciousness from anesthesia.

Hyperbaric Oxygen Treatment in Spinal Cord Injury Recovery: Profiling Long Noncoding RNAs.

STUDY DESIGN: A functional, transcriptome, and long noncoding RNAs (lncRNAs) expression analysis in the spinal cord of mice after hyperbaric oxygen (HBO) treatment. OBJECTIVE: We aimed to explore the mechanism by which HBO treats spinal cord injury (SCI) at the level of lncRNAs. SUMMARY OF BACKGROUND DATA: Immense amounts of research have established that HBO treatment promotes the recovery of neurological function after SCI. The mechanism of action remains to be clarified. METHODS: High-throughput RNA sequencing, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were used to profile lncRNA expression and analyze biological function in the spinal cords of mice from sham-operated, SCI, and HBO-treated groups. The differential expression of lncRNA between the groups was assessed using real-time quantitative polymerase chain reaction. RESULTS: Differential expression across 577 lncRNAs was identified among the three groups. GO analysis showed that free ubiquitin chain polymerization, ubiquitin homeostasis, DNA replication, synthesis of RNA primer, single-stranded telomeric DNA binding, and alpha-amylase activity were significantly enriched. Kyoto Encyclopedia of Genes and Genomes enrichment analysis displayed that vitamin B6 metabolism, one carbon pool by folate, DNA replication, lysine degradation, beta-alanine metabolism, fanconi anemia pathway, and Notch signal pathway were the main pathways with enrichment significance. LncRNAs NONMMUT 092674.1, NONMMUT042986.2, and NONMMUT018850.2 showed significantly different expression between the SCI and the other two groups (P<0.05, <0.01). CONCLUSIONS: This study is the first to determine the expression profiles of lncRNAs in the injured spinal cord after HBO treatment. We identified several important dysregulated lncRNAs in this setting. These results help us better understand the mechanism by which HBO treats SCI and provide new potential therapeutic targets for SCI.

A Phytophthora infestans RXLR effector targets a potato ubiquitin-like domain-containing protein to inhibit the proteasome activity and hamper plant immunity.

Ubiquitin-like domain-containing proteins (UDPs) are involved in the ubiquitin-proteasome system because of their ability to interact with the 26S proteasome. Here, we identified potato StUDP as a target of the Phytophthora infestans RXLR effector Pi06432 (PITG_06432), which supresses the salicylic acid (SA)-related immune pathway. By overexpressing and silencing of StUDP in potato, we show that StUDP negatively regulates plant immunity against P. infestans. StUDP interacts with, and destabilizes, the 26S proteasome subunit that is referred to as REGULATORY PARTICLE TRIPLE-A ATP-ASE (RPT) subunit StRPT3b. This destabilization represses the proteasome activity. Proteomic analysis and Western blotting show that StUDP decreases the stability of the master transcription factor SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) in SA biosynthesis. StUDP negatively regulates the SA signalling pathway by repressing the proteasome activity and destabilizing StSARD1, leading to a decreased expression of the SARD1-targeted gene ISOCHORISMATE SYNTHASE 1 and thereby a decrease in SA content. Pi06432 stabilizes StUDP, and it depends on StUDP to destabilize StRPT3b and thereby supress the proteasome activity. Our study reveals that the P. infestans effector Pi06432 targets StUDP to hamper the homeostasis of the proteasome by the degradation of the proteasome subunit StRPT3b and thereby suppresses SA-related immunity.

Short-term exposure to an obesogenic diet causes dynamic dysregulation of proteasome-mediated protein degradation in the hypothalamus of female rats.

OBJECTIVES: Previous work has shown that exposure to a high fat diet dysregulates the protein degradation process in the hypothalamus of male rodents. However, whether this occurs in a sex-independent manner is unknown. The objective of this study was to determine the effects of a short-term obesogenic diet on the ubiquitin-proteasome mediated protein degradation process in the hypothalamus of female rats. METHODS: We fed young adult female rats a high fat diet or standard rat chow for 7 weeks. At the end of the 7th week, animals were euthanized and hypothalamus nuclear and cytoplasmic fractions were collected. Proteasome activity and degradation-specific (K48) ubiquitin signaling were assessed. Additionally, we transfected female rats with CRISPR-dCas9-VP64 plasmids in the hypothalamus prior to exposure to the high fat diet in order to increase proteasome activity and determine the role of reduced proteasome function on weight gain from the obesogenic diet. RESULTS: We found that across the diet period, females gained weight significantly faster on the high fat diet than controls and showed dynamic downregulation of proteasome activity, decreases in proteasome subunit expression and an accumulation of degradation-specific K48 polyubiquitinated proteins in the hypothalamus. Notably, while our CRISPR-dCas9 manipulation was able to selectively increase some forms of proteasome activity, it was unable to prevent diet-induced proteasome downregulation or abnormal weight gain. CONCLUSIONS: Collectively, these results reveal that acute exposure to an obesogenic diet causes reductions in the protein degradation process in the hypothalamus of females.

Astaxanthin Inhibits Oxidative Stress-Induced Ku Protein Degradation and Apoptosis in Gastric Epithelial Cells.

Oxidative stress induces DNA damage which can be repaired by DNA repair proteins, such as Ku70/80. Excess reactive oxygen species (ROS) stimulate the activation of caspase-3, which degrades Ku 70/80. Cells with decreased Ku protein levels undergo apoptosis. Astaxanthin exerts antioxidant activity by inducing the expression of catalase, an antioxidant enzyme, in gastric epithelial cells. Therefore, astaxanthin may inhibit oxidative stress-induced DNA damage by preventing Ku protein degradation and thereby suppressing apoptosis. Ku proteins can be degraded via ubiquitination and neddylation which adds ubiquitin-like protein to substrate proteins. We aimed to determine whether oxidative stress decreases Ku70/80 expression through the ubiquitin-proteasome pathway to induce apoptosis and whether astaxanthin inhibits oxidative stress-induced changes in gastric epithelial AGS cells. We induced oxidative stress caused by the treatment of ß-D-glucose (G) and glucose oxidase (GO) in the cells. As a result, the G/GO treatment increased ROS levels, decreased nuclear Ku protein levels and Ku-DNA-binding activity, and induced the ubiquitination of Ku80. G/GO increased the DNA damage marker levels (γ-H2AX; DNA fragmentation) and apoptosis marker annexin V-positive cells and cell death. Astaxanthin inhibited G/GO-induced alterations, including Ku degradation in AGS cells. MLN4924, a neddylation inhibitor, and MG132, a proteasome inhibitor, suppressed G/GO-mediated DNA fragmentation and decreased cell viability. These results indicated that G/GO-induced oxidative stress causes Ku protein loss through the ubiquitin-proteasome pathway, resulting in DNA fragmentation and apoptotic cell death. Astaxanthin inhibited oxidative stress-mediated apoptosis via the reduction of ROS levels and inhibition of Ku protein degradation. In conclusion, dietary astaxanthin supplementation or astaxanthin-rich food consumption may be effective for preventing or delaying oxidative stress-mediated cell damage by suppressing Ku protein loss and apoptosis in gastric epithelial cells.

Virtual Drug Repositioning as a Tool to Identify Natural Small Molecules That Synergize with Lumacaftor in F508del-CFTR Binding and Rescuing.

Cystic fibrosis is a hereditary disease mainly caused by the deletion of the Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. Cystic fibrosis remains a potentially fatal disease, but it has become treatable as a chronic condition due to some CFTR-rescuing drugs that, when used in combination, increase in their therapeutic effect due to a synergic action. Also, dietary supplementation of natural compounds in combination with approved drugs could represent a promising strategy to further alleviate cystic fibrosis symptoms. On these bases, we screened by in silico drug repositioning 846 small synthetic or natural compounds from the AIFA database to evaluate their capacity to interact with the highly druggable lumacaftor binding site of F508del-CFTR. Among the identified hits, nicotinamide (NAM) was predicted to accommodate into the lumacaftor binding region of F508del-CFTR without competing against the drug but rather stabilizing its binding. The effective capacity of NAM to bind F508del-CFTR in a lumacaftor-uncompetitive manner was then validated experimentally by surface plasmon resonance analysis. Finally, the capacity of NAM to synergize with lumacaftor increasing its CFTR-rescuing activity was demonstrated in cell-based assays. This study suggests the possible identification of natural small molecules devoid of side effects and endowed with the capacity to synergize with drugs currently employed for the treatment of cystic fibrosis, which hopefully will increase the therapeutic efficacy with lower doses.

Study on the Effects of Different Doses of Dahuang Zhechong Pills on the Ubiquitin Proteasome Pathway/Nuclear Factor-κB in Rats with Atherosclerosis and Its Mechanism.

In order to investigate the effects of different doses of Dahuang Zhechong pills on the ubiquitin proteasome pathway/nuclear factor-κB (UPP-NF-κB) in rats with atherosclerosis (AS), 58-week-old male Wistar rats were selected and randomly divided into the normal group, model group, control group, low-dose group, and high-dose group. The model group and the drug group are given intraperitoneal injections of vitamins, and the model group and the drug group are given a high-fat diet. Rats in the low-dose group and high-dose group are given low-dose and high-dose Dahuang Zhechong pill lavage solution, respectively. Besides, the control group is given simvastatin solution by gavage, and intervention is performed once a day for 12 weeks. Ubiquitin (Ub) protein expression, ubiquitin activase (UBE1), nuclear factor-κB, nuclear inhibitory factor-κB (IκB) gene expression, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and serum tumor necrosis factor-α (TNF-α) are compared. The experimental result shows that Dahuang Zhechong pills can reduce inflammation and prevent and treat AS by blocking the activation of the UPP/NF-κB signaling pathway and can be used as a proteasome inhibitor in the clinical treatment of AS.

Tollip suppresses MyD88-mediated NF-κB activation by enhancing MyD88 ubiquitination levels in large yellow croaker (Larimichthys crocea).

Toll-interacting protein (Tollip) plays an important role in the innate immune response by negative regulation of the TLR-IL-1R signaling pathway. MyD88 serves as a universal adaptor in TLR-mediated NF-κB activation. However, the regulation mechanisms of Tollip in piscine MyD88-mediated NF-κB activation is largely unknown. In the present study, the cDNA sequence of LcTollip was identified from the large yellow croaker (Larimichthys crocea). The putative LcTollip protein encoded 275 amino acid residues, containing a N-terminal TBD domain, a central C2 domain, and a C-terminal CUE domain. Quantitative PCR showed that the most predominant constitutive expression of LcTollip was detected in spleen. In addition, LcTollip transcripts enhanced significantly after LPS and poly I:C challenge (P < 0.05). Cellular localization revealed that LcTollip existed in the cytoplasm and nucleus. Furthermore, the overexpression plasmids of wild type LcTollip as well as its six domain truncated mutants of LcTollip were constructed by overlap PCR. Dual luciferase analysis showed that NF-κB activation could not be induced by overexpression of LcTollip or its domain truncated mutants alone. However, the LcMyD88-induced-NF-κB activation was significantly suppressed by overexpression with LcTollip, and the truncated mutants LcTollip-ΔTBD, LcTollip-ΔC2, LcTollip-ΔCUE and LcTollip-ΔTBDΔCUE while not by LcTollip-ΔLR and LcTollip-ΔTBDΔC2. Moreover, co-immunoprecipitation (Co-IP) assay revealed that the interaction between LcTollip and LcMyD88 was through CUE domain. More interesting, IP and immunoblotting examination of HEK293T cells co-transfected with LcMyD88, LcTollip and HA-ubiquitin showed that LcMyD88 induced a dose-dependent de-ubiquitination of LcTollip while LcTollip enhanced a dose-dependent ubiquitination of LcMyD88. However, protein degradation investigation displayed that the proteolysis and ubiquitination of LcMyD88 were not connected. Our findings suggested that the LcTollip might involve in negative regulation TLR pathway by suppressing LcMyD88-mediated immune activation and improving the ubiquitination level of LcMyD88.

Synergistic antitumor effects of compound-composed optimal formula from Aidi injection on hepatocellular carcinoma and colorectal cancer.

BACKGROUND: Traditional Chinese medicine formula (TCMF) possesses unique advantages in the prevention and treatment of malignant tumors such as hepatocellular carcinoma (HCC) and colorectal cancer (CRC). However, the unclear chemical composition and mechanism lead to its unstable efficacy and adverse reactions occurring frequently, especially injection. We previously proposed the research idea and strategy for compound-composed Chinese medicine formula (CCMF). PURPOSE: A demonstration study was performed through screening of the compound-composed optimal formula (COF) from Aidi injection, confirmation of the synergistic effect, and exploration of the related mechanism in the treatment of HCC and CRC. METHOD: The feedback system control (FSC) technique was applied to screening of COF. CCK-8 and calcein-AM/PI assays were performed to evaluate cell proliferation. Cell apoptosis was assessed using flow cytometry and DAPI staining. JC-1 probe and mitochondrial staining were employed to detect mitochondrial membrane potential (MMP) and the release of cytochrome c into cytoplasm, respective. Quantitative proteomics, drug affinity responsive target stability (DARTS) assay, bioinformatics, and molecular docking were carried out to explore the targets of the compounds and the synergistic mechanism involved. RESULTS: COF was obtained from Aidi injection, which comprises cantharidin (CAN): calycosin-7-O-ß-D-glucoside (CAG): ginsenoside Rc: ginsenoside Rd = 1:12:12:8 (molar ratio). The monarch drug CAN in combination with minister medicines consisting of CAG, Rc and Rd (abbr. TD) displayed evidently synergistic effect, which inhibited cell viability, increased dead cell number, induced apoptosis, reduced MMP, promoted cytochrome c leakage of HCC and CRC cells, and suppressed the increases of tumor volume and weight in HCC and CRC bearing nude mice. TD probably antagonized CAN enhanced activity of the ubiquitin proteasome system (UPS) to depress the degradation of cytotoxic proteins through binding to ubiquitin proteasome, thus exerting the synergistic effect with CAN activated protein phosphatase 2A (PP2A) to activate the mitochondrial apoptosis pathway. In addition, the CAN enhanced protein expression of UPS was also observed for the first time. CONCLUSION: CAN and TD exert synergism through activation of PP2A and inhibition of UPS. It makes sense to elucidate the scientific nature of the compatibility theory of TCMF based on CCMF, which will be an important research direction of the modernization of traditional Chinese medicines.

Signal pathways in the treatment of Alzheimer's disease with traditional Chinese medicine.

AIM OF THE REVIEW: This study aimed to reveal the classical signal pathways and important potential targets of traditional Chinese medicine (TCM) for treating Alzheimer's disease (AD), and provide support for further investigation on TCM and its active ingredients. MATERIALS AND METHODS: Literature survey was conducted using PubMed, Web of Science, Google Scholar, CNKI, and other databases, with "Alzheimer's disease," "traditional Chinese medicine," "medicinal herb," "Chinese herb," and "natural plant" as the primary keywords. RESULTS: TCM could modulate signal pathways related to AD pathological progression, including NF-κB, Nrf2, JAK/STAT, ubiquitin-proteasome pathway, autophagy-lysosome pathway-related AMPK/mTOR, GSK-3/mTOR, and PI3K/Akt/mTOR, as well as SIRT1 and PPARα pathway. It could regulate crosstalk between pathways through a multitarget, thus maintaining chronic inflammatory interaction balance, inhibiting oxidative stress damage, regulating ubiquitin-proteasome system function, modulating autophagy, and eventually improving cognitive impairment in patients with AD. CONCLUSION: TCM could be multilevel, multitargeted, and multifaceted to prevent and treat AD. In-depth research on the prevention and treatment of AD with TCM could provide new ideas for exploring the pathogenesis of AD and developing new anti-AD drugs.

New Strains of Akkermansia muciniphila and Faecalibacterium prausnitzii are Effective for Improving the Muscle Strength of Mice with Immobilization-Induced Muscular Atrophy.

Muscular atrophy is a muscle disease in which muscle mass and strength decrease due to aging, injury, metabolic disorders, or chronic conditions. Proteins in muscle tissue are degraded by the ubiquitin-proteasome pathway, and atrophy accelerates this pathway. Akkermansia muciniphila and Faecalibacterium prausnitzii strains are effective agents against metabolic and inflammatory diseases in next-generation probiotic research. In this study, we evaluated the efficacy of A. muciniphila strain EB-AMDK19 and F. prausnitzii strain EB-FPDK11 in a mouse model of muscular atrophy, since atrophy inhibits energy metabolism and immune activation. After oral administration of each strain for 4 weeks, the hind legs of the mice were fixed with a plaster cast to immobilize them for a week. As a result, the administration of EB-AMDK19 and EB-FPDK11 strains improved grip strength but did not increase muscle mass. At the molecular level, A. muciniphila and F. prausnitzii treatments decreased the expression levels of ubiquitin-proteasome genes, atrogin-1, MuRF, and cathepsin L. They increased the expression level of the mitochondrial biogenesis regulatory gene, PGC-1α. The effect of the strains was confirmed by a decrease in myostatin. Furthermore, A. muciniphila and F. prausnitzii modulated the immune function by enhancing ZO-1 and inhibiting IL-6. In particular, EB-AMDK19 promoted the expression of IL-10, an anti-inflammatory cytokine. These results suggest that A. muciniphila and F. prausnitzii may have beneficial effects on muscular atrophy, verified by newly isolated EB-AMDK19 and EB-FPDK11 as potential next-generation probiotics.

Protective effect of resveratrol on rat cardiomyocyte H9C2 cells injured by hypoxia/reoxygenation by regulating mitochondrial autophagy PTEN-induced putative kinase protein 1/Parkinson disease protein 2 signaling pathway.

OBJECTIVE: To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/ reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2 (PINK1/PARKIN) signaling pathway. METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide was used to detect the effect of resveratrol on the viability of H9C2 cells; the hypoxia/ reoxygenation (H/R) model was established in tri-gas incubator; 2', 7'-Dichlorofluorescin diacetate staining was used to measure the content of reactive oxygen species (ROS); the changes of mitochondrial membrane potential was determined by 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide staining; the changes of mitochondrial respiratory chain complex activity was evaluated by enzyme activity kits; flow cytometry was used to detect the ratio of apoptotic cells; transmission electron microscope was used to observe the ultrastructure of H9C2 cells; Western blot was used to detect the protein changes of mitochondrial 20 kDa outer membrane protein (TOM20), translocase of inner mitochondrial membrane 23 (TIM23), presenilins associated rhomboid-like protein (PARL), PINK1, PARKIN and mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), phosphotyrosine independent ligand for the Lck SH2 domain of 62 kDa (P62), microtubule-associated protein 1 light chain 3 beta (LC3B); the mRNA levels of PINK1 and PARKIN was detected by quantitative polymerase chain reaction; immunoprecipitation assay was used to detect the interaction between PARKIN and Ubiquitin. RESULTS: Resveratrol could inhibit the proliferation of H9C2 cells in a time- and concentration- dependent manner; however, pretreatment with low cytotoxic resveratrol could reduce the H/R-induced increase in cellular ROS levels, alleviate the loss of mitochondrial membrane potential induced by H/R, inhibit H/R-induced apoptosis of H9C2 cells, and protect the mitochondrial structure and respiratory chain of H9C2 cells from H/R damage. Resveratrol could further increase the levels of p62, PINK1, PARKIN protein, the expression of PINK1, PARKIN mRNA and the ratio of LC3BⅡ/LC3BⅠin H/R-induced H9C2 cells, inhibit the interaction between PARKIN and Ubiquitin in H/R-induced H9C2 cells, and further reduce the expression of TOM20,TIM23, PARL, Mfn1 and Mfn2 protein in H/R-induced H9C2 cells. The effect of resveratrol is consistent with that of autophagy activator on H/R-induced H9C2 cells. CONCLUSIONS: Resveratrol can protect H9C2 cells from H/R injury, which may be related to resveratrol promoting mitochondrial autophagy by activating PINK1/PARKIN signaling pathway.