Picornavirus VP3 protein induces autophagy through the TP53-BAD-BAX axis to promote viral replication.

Macroautophagy/autophagy and apoptosis are pivotal interconnected host cell responses to viral infection, including picornaviruses. Here, the VP3 proteins of picornaviruses were determined to trigger autophagy, with the autophagic flux being triggered by the TP53-BAD-BAX axis. Using foot-and-mouth disease virus (FMDV) as a model system, we unraveled a novel mechanism of how picornavirus hijacks autophagy to bolster viral replication and enhance pathogenesis. FMDV infection induced both autophagy and apoptosis in vivo and in vitro. FMDV VP3 protein facilitated the phosphorylation and translocation of TP53 from the nucleus into the mitochondria, resulting in BAD-mediated apoptosis and BECN1-mediated autophagy. The amino acid Gly129 in VP3 is essential for its interaction with TP53, and crucial for induction of autophagy and apoptosis. VP3-induced autophagy and apoptosis are both essential for FMDV replication, while, autophagy plays a more important role in VP3-mediated pathogenesis. Mutation of Gly129 to Ala129 in VP3 abrogated the autophagic regulatory function of VP3, which significantly decreased the viral replication and pathogenesis of FMDV. This suggested that VP3-induced autophagy benefits viral replication and pathogenesis. Importantly, this Gly is conserved and showed a common function in various picornaviruses. This study provides insight for developing broad-spectrum antivirals and genetic engineering attenuated vaccines against picornaviruses.Abbreviations: 3-MA, 3-methyladenine; ATG, autophagy related; BAD, BCL2 associated agonist of cell death; BAK1, BCL2 antagonist/killer 1; BAX, BCL2 associated X, apoptosis regulator; BBC3/PUMA, BCL2 binding component 3; BCL2, BCL2 apoptosis regulator; BID, BH3 interacting domain death agonist; BIP-V5, BAX inhibitor peptide V5; CFLAR/FLIP, CASP8 and FADD like apoptosis regulator; CPE, cytopathic effects; CQ, chloroquine; CV, coxsackievirus; DAPK, death associated protein kinase; DRAM, DNA damage regulated autophagy modulator; EV71, enterovirus 71; FMDV, foot-and-mouth disease virus; HAV, hepatitis A virus; KD, knockdown; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MOI, multiplicity of infection; MTOR, mechanistic target of rapamycin kinase; PML, promyelocytic leukemia; PV, poliovirus; SVA, Seneca Valley virus; TCID50, 50% tissue culture infectious doses; TOR, target of rapamycin. TP53/p53, tumor protein p53; WCL, whole-cell lysate.

Kellerin alleviates cerebral ischemic injury by inhibiting ferroptosis via targeting Akt-mediated transcriptional activation of Nrf2.

BACKGROUND: Ischemic stroke (IS) is characterized as a detrimental cerebrovascular disease with high mortality and disability. Ferroptosis is a novel mechanism involved in neuronal death. There is a close connection between IS and ferroptosis, and inhibiting ferroptosis may provide an effective strategy for treating IS. Our previous investigations have discovered that kellerin, the active compound of Ferula sinkiangensis K. M. Shen, possesses the capability to shield against cerebral ischemia injury. PURPOSE: Our objective is to clarify the relationship between the neuroprotective properties of kellerin against IS and its ability to modulate ferroptosis, and investigate the underlying regulatory pathway. STUDY DESIGN: We investigated the impact and mechanism of kellerin in C57BL/6 mice underwent middle cerebral artery occlusion/reperfusion (MCAO/R) as well as SH-SY5Y cells exposed to oxygen-glucose deprivation/ re-oxygenation (OGD/R). METHODS: The roles of kellerin on neurological severity, cerebral infarction and edema were investigated in vivo. The regulatory impacts of kellerin on ferroptosis, mitochondrial damage and Akt/Nrf2 pathway were explored. Molecular docking combined with drug affinity responsive target stability assay (DARTS) and cellular thermal shift assay (CETSA) were performed to analyze the potential target proteins for kellerin. RESULTS: Kellerin protected against IS and inhibited ferroptosis in vivo. Meanwhile, kellerin improved the neuronal damage caused by OGD/R and suppressed ferroptosis by inhibiting the production of mitochondrial ROS in vitro. Further we found that kellerin directly interacted with Akt and enhanced its phosphorylation, leading to the increase of Nrf2 nuclear translocation and its downstream antioxidant genes expression. Moreover, kellerin's inhibitory effect on ferroptosis and mitochondrial ROS release was eliminated by inhibiting Akt/Nrf2 pathway. CONCLUSIONS: Our study firstly demonstrates that the neuroprotective properties of kellerin against IS are related to suppressing ferroptosis through inhibiting the production of mitochondrial ROS, in which its modulation on Akt-mediated transcriptional activation of Nrf2 plays an important role. This finding shed light on the potential mechanism that kellerin exerts therapeutic effects in IS.

Novel Ziyuglycoside II derivatives inhibit MCF-7 cell proliferation via inducing apoptosis and autophagy.

A series of novel ziyuglycoside II derivatives were synthesized based on the classical 1,2,3-triazole moiety. Among the tested derivatives (Z-1 - Z-15), the compound Z-15 demonstrated the most potent antiproliferative effect on K562, MCF-7 and MV411 cell lines. Moreover, Z-15 did not show obvious cytotoxicity on MCF-10A cell, a human normal mammary epithelial cell. The cell colony formation assay showed that, compared to ziyuglycoside II and 5-fluorouracil, Z-15 could inhibit cell proliferation more robustly. Wound healing assays indicated that Z-15 could significantly inhibit MCF-7 cell migration. Further mechanistic research revealed that Z-15 induced mitochondrial-mediated apoptosis and autophagy in MCF-7 cell line in a dose-dependent manner.

[Role of podocyte injury signaling pathway in steroid-resistant nephrotic syndrome and research progress in traditional Chinese medicine intervention].

As one of the main diseases leading to end-stage renal disease, steroid-resistant nephrotic syndrome(SRNS) can cause serious complications such as infection. Without effective control, this disease can further lead to the malignant development of the renal function, bringing serious social and economic burdens. As previously reported, the formation of SRNS is mostly related to the podocyte injury in the body, i.e., the injury of glomerular visceral epithelial cells. Phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway, nuclear transcription factor-κB(NF-κB) signaling pathway, mammalian target of rapamycin(mTOR)/adenosine monophosphate(AMP)-activated protein kinase(AMPK), transforming growth factor(TGF)-ß1/Smads, and other signaling pathways are classical signaling pathways related to podocyte injury. By regulating the expression of signaling pathways, podocyte injury can be intervened to improve the adhesion between podocyte foot processes and glomerular basement membrane and promote the function of podocytes, thereby alleviating the clinical symptoms of SRNS. Through the literature review, traditional Chinese medicine(TCM) has unique advantages and an important role in intervening in podocyte injury. In the intervention in podocyte injury, TCM, by virtue of multi-target and multi-pathway role, can regulate and intervene in podocyte injury in many ways, alleviate the clinical symptoms of SRNS, and interfere with the progress of SRNS, reflecting the unique advantages of TCM. On the other hand, TCM can directly or indirectly inhibit podocyte injury by regulating the above signaling pathways, which can not only promote the effect of hormones and immunosuppressants and shorten the course of treatment, but also reduce the toxic and side effects caused by various hormones and immunosuppressants to exert the advantages of small side effects and low price of TCM. This article reviewed TCM in the treatment of SRNS by interfering with podocyte injury-related signaling pathways and is expected to provide a reference for the in-depth study of TCM in the treatment of SRNS, as well as a theoretical basis and a new direction for the clinical application of TCM to shorten the course of treatment of SRNS and delay the progression to end-stage renal disease.

Long non-coding RNA UCA1 regulates MPP+-induced neuronal damage through the miR-671-5p/KPNA4 pathway in SK-N-SH cells.

Parkinson's disease (PD) is an age-related neurodegenerative disease. Long non-coding RNA urothelial carcinoma-associated 1 (UCA1) is involved in the pathogenesis of PD. However, the pathogenesis of PD regulated by UCA1 has not been fully explained. We used 1-Methyl-4-phenylpyridinium (MPP+)-induced SK-N-SH cells for functional analysis. Expression levels of UCA1, microRNA (miR)-671-5p, and KPNA4 (karyopherin subunit alpha 4) mRNA were detected using quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis were analyzed using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) or flow cytometry assays. Some protein levels were measured by western blotting. The levels of pro-inflammatory cytokines were tested by ELISA (enzyme-linked immunosorbent assay). The levels of LDH (lactate dehydrogenase), MDA (malondialdehyde), and SOD (superoxide dismutase) were measured using corresponding kits. The relationship between UCA1 or KPNA4 and miR-671-5p was verified by dual-luciferase reporter assay and/or RNA immunoprecipitation (RIP) assay. MPP+ induced UCA1 expression in SK-N-SH cells in a concentration-dependent manner or time-dependent manner. UCA1 knockdown reduced MPP+-induced apoptosis, inflammation, and oxidative stress in SK-N-SH cells. MiR-671-5p was downregulated while KPNA4 was upregulated in MPP+-treated SK-N-SH cells. UCA1 sponged miR-671-5p to regulate KPNA4 expression. MiR-671-5p inhibition counteracted UCA1 knockdown-mediated influence on apoptosis, inflammation, and oxidative stress of MPP+-induced SK-N-SH cells. KPNA4 overexpression offset the inhibitory influence of miR-671-5p mimic on apoptosis, inflammation, and oxidative stress of MPP+-treated SK-N-SH cells. UCA1 inhibition reduced MPP+-induced neuronal damage through the miR-671-5p/KPNA4 pathway in SK-N-SH cells, providing a novel mechanism to understand the pathogenesis of PD.

Long term neuropsychiatric consequences in COVID-19 survivors: Cognitive impairment and inflammatory underpinnings fifteen months after discharge.

BACKGROUND: Emerging evidence shows that cognitive dysfunction may occur following coronavirus disease 19 (COVID-19) infection which is one of the most common symptoms reported in researches of "Long COVID". Several inflammatory markers are known to be elevated in COVID-19 survivors and the relationship between long-term inflammation changes and cognitive function remains unknown. METHODS: We assessed cognitive function and neuropsychiatric symptoms of 66 COVID-19 survivors and 79 healthy controls (HCs) matched with sex, age, and education level using a digital, gamified cognitive function evaluation tool and questionnaires at 15 months after discharge. Venous blood samples were collected to measure cytokine levels. We performed correlation analyses and multiple linear regression analysis to identify the factors potentially related to cognitive function. RESULTS: The COVID-19 survivors performed less well on the Trails (p = 0.047) than the HCs, but most of them did not report subjective neuropsychiatric symptoms. Intensive care unit experience (ß = -2.247, p < 0.0001) and self-perceived disease severity (ß = -1.522, p = 0.007) were positively correlated, whereas years of education (ß = 0.098, p = 0.013) was negatively associated with the performance on the Trails. Moreover, the abnormally elevated TNF-α levels (r = -0.19, p = 0.040) were negatively correlated with performance on the Trails in COVID-19 group. CONCLUSION: Our findings suggest that COVID-19 survivors show long-term cognitive impairment in executive function, even at 15 months after discharge. Serum TNF-α levels may be an underlying mechanism of long-term cognitive impairment in patients recovering from COVID-19.

Selective bromodomain and extra-terminal bromodomain inhibitor inactivates macrophages and hepatic stellate cells to inhibit liver inflammation and fibrosis.

Liver fibrosis occurs following inflammation triggered by the integrated actions of activated liver-resident macrophages (Kupffer cells) and hepatic stellate cells (HSCs), and the multiplicity of these mechanisms complicates drug therapy. Here, we demonstrate that the selective bromodomain and extra-terminal (BET) bromodomain inhibitor compound38 can block both the Janus kinase-signal transducer and activator of transcription and mitogen-activated protein kinase signaling pathways in macrophages, which decreased their secretion of proinflammatory cytokines in a dose-dependent manner. The inactivation of macrophages attenuated lipopolysaccharide-induced injurious inflammation concurrent with a reduction in F4/80+ cells, proinflammatory cytokine levels, and neutrophil infiltration. Moreover, compound 38 inhibited the Wnt/ß-catenin and transforming growth factor-beta/SMAD signaling pathways to abolish the activation of HSCs. In vivo, compound 38 significantly decreased the collagen deposition and fibrotic area of a CCl4-induced liver fibrosis model, and restored the deficiency of activated HSCs and the upregulation of liver inflammation. These results highlight the potential role of compound 38 in treating liver fibrosis considering its simultaneous inhibitory effects on liver inflammation and related fibrosis.

Yixin Ningshen Tablet Alleviates Comorbidity of Myocardial Infarction and Depression by Enhancing Myocardial Energy Metabolism and Increasing Availability of Monoamine Neurotransmitter.

OBJECTIVE: To investigate the therapeutic effect of Yixin Ningshen Tablet (YXNS) on comorbidity of myocardial infarction (MI) and depression in rats and explore the underlying mechanism. METHODS: The Sprague-Dawley rats were randomly divided into 5 groups with 7 rats in each group according to their weights, including control, model, fluoxetine (FLXT, 10 mg/kg), low-dose YXNS (LYXNS, 100 mg/kg), and high-dose YXNS (HYXNS, 300 mg/kg) groups. All rats were pretreated with corresponding drugs for 12 weeks. The rat model of MI and depression was constructed by ligation of left anterior descending coronary artery and chronic mild stress stimulation. The echocardiography, sucrose preference test, open field test, and forced swim test were performed. Myocardial infarction (MI) area and myocardial apoptosis was also detected. Serum levels of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α (TNF-α), 5-hydroxytryptamine (5-HT), adrenocorticotrophic hormone (ACTH), corticosterone (CORT), and norepinephrine (NE) were determined by enzyme linked immunosorbent assay. The proteins of adenosine 5'-monophosphate -activated protein kinase (AMPK), p-AMPK, peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), and nuclear respiratory factor 1 (NRF1) in heart were detected by Western blot analysis. The expression levels of TNF-α, IL-6, indoleamine 2,3-dioxygenase (IDO1), kynurenine 3-monooxygenase (KMO), and kynureninase (KYNU) in hippocampus were detected by real-time quantitative polymerase chain reaction. RESULTS: Compared with the model group, the cardiac function of rats treated with YXNS improved significantly (P<0.01). Meanwhile, YXNS effectively reduced MI size and cardiomyocytes apoptosis of rats (P<0.01 or P<0.05), promoted AMPK phosphorylation, and increased PGC-1α protein expression (P<0.01 or P<0.05). HYXNS significantly increased locomotor activity of rats, decreased the levels of TNF-α, IL-6 and IL-1ß, and increased the serum levels of 5-HT, NE, ACTH, and CORT (all P<0.05). Moreover, HYXNS decreased the mRNA expressions of IDO1, KMO and KYNU (P<0.05). CONCLUSIONS: YXNS can relieve MI by enhancing myocardial energy metabolism. Meanwhile, YXNS can alleviate depression by resisting inflammation and increasing availability of monoamine neurotransmitters. It may be used as a potential drug to treat comorbidity of MI and depression.

[Concentration Analysis and Health Risk Assessment of Air Pollutants in Newly Decorated Public Places in Xi'an].

In order to understand the levels of indoor air pollution and health risks in public places, our research group conducted air quality monitoring and human health risk assessments for five types of public places (offices, classrooms, laboratories, banks, and hospitals) in Xi'an City from December 2017 to July 2020. The test items included formaldehyde, benzene, toluene, xylene, n-butyl acetate, ethylbenzene, styrene, n-undecane, and total volatile organic compounds (TVOC). The results showed that formaldehyde had the highest exceedance rate (59.4%), followed by toluene, TVOC, benzene, and xylene. Among the five types of public places, hospitals had the highest rate of pollutants exceeding the standard (46.7%), and the main pollutants exceeding the standard were formaldehyde, benzene, and toluene. The results showed that the concentrations of formaldehyde and TVOC were positively correlated with temperature and humidity. The health risk assessment results showed that there were carcinogenic risks of formaldehyde and benzene in different places; people working in banks had a higher risk of formaldehyde carcinogenesis, and those working in hospitals had a higher risk of benzene carcinogenesis. This study provides a reference for the level of indoor air pollution in public places in Xi'an City, and is of great significance to the health risk research of related populations.

Anti-proliferation and apoptosis-inducing effects of sodium aescinate on retinoblastoma Y79 cells.

AIM: To investigate the anti-proliferation and apoptosis-inducing effects of sodium aescinate (SA) on retinoblastoma Y79 cells and its mechanism. METHODS: Y79 cells were cultured at different drug concentrations for different periods of time (24, 48, and 72h). The inhibitory effect of SA on proliferation of Y79 cells was detected by the cell counting kit-8 (CCK-8) assay, and the morphology of Y79 cells in each group was observed under an inverted microscope. An IC50 of 48h was selected for subsequent experiments. After pretreatment with SA for 24 and 48h, cellular DNA distribution and apoptosis were detected by flow cytometry. Real-time qunatitative polymerase chain reaction (RT-qPCR) and Western blot were used to assess changes in related genes (CDK1, CyclinB1, Bax, Bcl-2, caspase-9, caspase-8, and caspase-3). RESULTS: SA inhibited proliferation and induced apoptosis of Y79 cells in a time-dependent and concentration-dependent manner. Following its intervention in the cell cycle pathway, SA can inhibit the expression of CDK1 and CyclinB1 at the mRNA and protein levels, and block cells in the G2/M phase. In caspase-related apoptotic pathways, up-regulation of Bax and down-regulation of Bcl-2 caused caspase-9 to self-cleave and further activate caspase-3. What's more, the caspase-8-mediated extrinsic apoptosis pathway was activated, and the activated caspase-8 was released into the cytoplasm to activate caspase-3, which as a member of the downstream apoptotic effect group, initiates a caspase-cascade reaction that induces cell apoptosis. CONCLUSION: SA inhibits the proliferation of Y79 cells by arresting the cell cycle at the G2/M phase, and induces apoptosis via the caspase-related apoptosis pathway, indicating that SA may have promising potential as a chemotherapeutic drug.

Sorting Nexin 10 Mediates Metabolic Reprogramming of Macrophages in Atherosclerosis Through the Lyn-Dependent TFEB Signaling Pathway.

RATIONALE: SNX10 (sorting nexin 10) has been reported to play a critical role in regulating macrophage function and lipid metabolism. OBJECTIVE: To investigate the precise role of SNX10 in atherosclerotic diseases and the underlying mechanisms. METHODS AND RESULTS: SNX10 expression was compared between human healthy vessels and carotid atherosclerotic plaques. Myeloid cell-specific SNX10 knockdown mice were crossed onto the APOE-/- (apolipoprotein E) background and atherogenesis (high-cholesterol diet-induced) was monitored for 16 weeks. We found that SNX10 expression was increased in atherosclerotic lesions of aortic specimens from humans and APOE-/- mice. Myeloid cell-specific SNX10 deficiency (Δ knockout [KO]) attenuated atherosclerosis progression in APOE-/- mice. The population of anti-inflammatory monocytes/macrophages was increased in the peripheral blood and atherosclerotic lesions of ΔKO mice. In vitro experiments showed that SNX10 deficiency-inhibited foam cell formation through interrupting the internalization of CD36, which requires the interaction of SNX10 and Lyn-AKT (protein kinase B). The reduced Lyn-AKT activation by SNX10 deficiency promoted the nuclear translocation of TFEB (transcription factor EB), thereby enhanced lysosomal biogenesis and LAL (lysosomal acid lipase) activity, resulting in an increase of free fatty acids to fuel mitochondrial fatty acid oxidation. This further promoted the reprogramming of macrophages and shifted toward the anti-inflammatory phenotype. CONCLUSIONS: Our data demonstrate for the first time that SNX10 plays a crucial role in diet-induced atherogenesis via the previously unknown link between the Lyn-Akt-TFEB signaling pathway and macrophage reprogramming, suggest that SNX10 may be a potentially promising therapeutic target for atherosclerosis treatment.

MicroRNA-1249 targets four-jointed box kinase 1 and reduces cell proliferation, migration and invasion of colon adenocarcinoma.

BACKGROUND: MiR-1249 was demonstrated to be dysregulated and related to prognosis in cancers. It has been reported to be significantly down-regulated in colon adenocarcinoma (COAD). The present study aimed to explore the clinical value and biological roles of miR-1249 in the progression of COAD. METHODS: miRWalk was applied to predict potential targets of miR-1249. We investigated the expression patterns of miR-1249 and its potential target Four-Jointed Box Kinase 1 (FJX1) in COAD samples from The Cancer Genome Atlas (TCGA) or ONCOMINE database. Kaplan-Meier with a log-rank test was used to reveal the relationship between overall survival (OS) and miR-1249/FJX1. The predictive ability of miR-1249/FJX1 was investigated using univariate and multivariate Cox regression models. CCK-8 and Transwell assays were performed to determine whether miR-1249 was connected with cell viability, migration and invasion. A luciferase reporter assay was applied to verify the association of miR-1249 and FJX1 as its predicted target gene. RESULTS: We predicted and confirmed FJX1 to be a target gene of miR-1249. MiR-1249 was down-regulated in COAD samples and cell lines. Univariate and multivariate analysis showed that the expression of FJX1 could be regarded as independent predictor for COAD. Moreover, miR-1249 and FJX1 were respectively the indicators of favorable and poor OS. MiR-1249 over-expression repressed cell growth, migration and invasion. Overexpression of FJX1 in cells treated with miR-1249 mimic abolished the inhibitory effect of miR-1249 on cell growth, migration and invasion. CONCLUSIONS: miR-1249 exerts a suppressive effect on cell proliferation, migration and invasion in COAD, which is possibly achieved via modulating FJX1.

Astragaloside IV reverses simvastatin-induced skeletal muscle injury by activating the AMPK-PGC-1α signalling pathway.

In this study, we investigated the effect of astragaloside IV on skeletal muscle energy metabolism disorder caused by statins and explored the possible mechanisms. High-fat diet-fed apolipoprotein E knockout (ApoE-/- ) mice performed aerobic exercise and were administered simvastatin, simvastatin + trimetazidine, or simvastatin + astragaloside IV by gavage. At the end of treatment, exercise performance was assessed by the hanging grid test, forelimb grip test, and running tolerance test. Moreover, plasma lipid and creatine kinase concentrations were measured. After sacrifice, the gastrocnemius muscle was used to assess muscle morphology, and energy metabolism was evaluated by determining the concentration of lactic acid and the storage capacity of adenosine triphosphate and glycogen. Mitochondrial function was assessed by measuring mitochondrial complex III and citrate synthase activity and membrane potential. In addition, oxidative stress was assessed by determining the level of hydrogen peroxide. Finally, using western blotting and reverse transcription polymerase chain reaction, we explored the mechanism of astragaloside IV in alleviating simvastatin-induced muscle injury. Our results demonstrated that astragaloside IV reversed simvastatin-induced muscle injury without affecting the lipid-lowering effect of simvastatin. Moreover, astragaloside IV promoted the phosphorylation of AMPK and activated PGC-1α, which upregulated the expression of NRF1 to enhance energy metabolism and inhibit skeletal muscle cell apoptosis.

Total Glucosides of Paeony protects against collagen-induced mouse arthritis via inhibiting follicular helper T cell differentiation.

BACKGROUND: The development of rheumatoid arthritis (RA) is related to germinal center (GC) response and autoreactive T cells, which mediate adaptive immunity and play an important role in stimulating the production of autoantibodies and pro-inflammatory cytokines by B cells and macrophages. Total Glucosides of Paeony (TGP) has anti-inflammatory, immunomodulatory and analgesic effects and is widely used to treat RA. However, few studies investigated whether the therapeutic effect of TGP is associated with the inhibition of autoimmune response. PURPOSE: The aim of this study was to investigate the effects and mechanisms of TGP on RA. STUDY DESIGN: Type II collagen-induced arthritis (CIA) mouse model was used, and TGP and paeoniflorin were intragastrically treated. METHODS: DBA/1 mice were divided into 5 groups: control, model, positive drug (paeoniflorin) and high- and low-dose TGP group. After 21 days of intragastric administration, the pathological change, inflammation expression and molecular mechanism of each group of mice were detected by Micro-CT, histochemical analysis, ELLSA, Western blot, RT-qPCR and flow cytometry. RESULTS: Our study found that TGP treatment effectively improved inflammation and joint destruction in CIA mice. It reduced the production of serum IgG2a and pro-inflammatory cytokines, including serum interleukin (IL)-21, tumor necrosis factor (TNF)-α and IL-6, and the phosphorylation of NF-κB p65 and STAT3 in a dose-dependent manner. More importantly, TGP could suppress the frequency of germinal center B cells and Tfh cells in the spleen. CONCLUSION: TGP can not only improve symptoms, but also inhibit bone destruction. The therapeutic effect of TGP on CIA is mainly achieved by inhibiting spleen Tfh cell differentiation and GC formation through STAT3 signaling pathway.

Nitazoxanide Inhibits Human Norovirus Replication and Synergizes with Ribavirin by Activation of Cellular Antiviral Response.

Norovirus is the main cause of viral gastroenteritis worldwide. Although norovirus gastroenteritis is self-limiting in immunocompetent individuals, chronic infections with debilitating and life-threatening complications occur in immunocompromised patients. Nitazoxanide (NTZ) has been used empirically in the clinic and has demonstrated effectiveness against norovirus gastroenteritis. In this study, we aimed at uncovering the antiviral potential and mechanisms of action of NTZ and its active metabolite, tizoxanide (TIZ), using a human norovirus (HuNV) replicon. NTZ and TIZ, collectively referred to as thiazolides (TZD), potently inhibited replication of HuNV and a norovirus surrogate, feline calicivirus. Mechanistic studies revealed that TZD activated cellular antiviral response and stimulated the expression of a subset of interferon-stimulated genes (ISGs), particularly interferon regulatory factor 1 (IRF-1), not only in a Huh7 cell-based HuNV replicon, but also in naive Huh7 and Caco-2 cells and novel human intestinal organoids. Overexpression of exogenous IRF-1 inhibited HuNV replication, whereas knockdown of IRF-1 largely attenuated the antiviral activity of TZD, suggesting that IRF-1 mediated TZD inhibition of HuNV. By using a Janus kinase (JAK) inhibitor, CP-690550, and a STAT1 knockout approach, we found that TZD induced antiviral response independently of the classical JAK-signal transducers and activators of transcription (JAK-STAT) pathway. Furthermore, TZD and ribavirin synergized to inhibit HuNV replication and completely depleted the replicons from host cells after long-term treatment. In summary, our results demonstrated that TZD combated HuNV replication through activation of cellular antiviral response, in particular by inducing a prominent antiviral effector, IRF-1. NTZ monotherapy or combination with ribavirin represent promising options for treating norovirus gastroenteritis, especially in immunocompromised patients.

6-Thioguanine inhibits rotavirus replication through suppression of Rac1 GDP/GTP cycling.

Rotavirus infection has emerged as an important cause of complications in organ transplantation recipients and might play a role in the pathogenesis of inflammatory bowel disease (IBD). 6-Thioguanine (6-TG) has been widely used as an immunosuppressive drug for organ recipients and treatment of IBD in the clinic. This study aims to investigate the effects and mode-of-action of 6-TG on rotavirus replication. Human intestinal Caco2 cell line, 3D model of human primary intestinal organoids, laboratory rotavirus strain (SA11) and patient-derived rotavirus isolates were used. We have demonstrated that 6-TG significantly inhibits rotavirus replication in these intestinal epithelium models. Importantly, gene knockdown or knockout of Rac1, the cellular target of 6-TG, significantly inhibited rotavirus replication, indicating the supportive role of Rac1 for rotavirus infection. We have further demonstrated that 6-TG can effectively inhibit the active form of Rac1 (GTP-Rac1), which essentially mediates the anti-rotavirus effect of 6-TG. Consistently, ectopic over-expression of GTP-Rac1 facilitates but an inactive Rac1 (N17) or a specific Rac1 inhibitor (NSC23766) inhibits rotavirus replication. In conclusion, we have identified 6-TG as an effective inhibitor of rotavirus replication via the inhibition of Rac1 activation. Thus, for transplantation patients or IBD patients infected with rotavirus or at risk of rotavirus infection, the choice of 6-TG as a treatment appears rational.

IRF-1, RIG-I and MDA5 display potent antiviral activities against norovirus coordinately induced by different types of interferons.

Norovirus represents the main cause of acute nonbacterial gastroenteritis worldwide. In immunocompromised patients, it bears high risk of causing chronic infection with significant morbidity and mortality. The lack of specific treatment prompts the development of anti-norovirus agents. In this study, we have investigated the role of interferon (IFN) response and evaluated antiviral activities of different IFNs against human norovirus (HuNoV) replication using a HuNoV replicon model. We found that HuNoV RNA replication was sensitive to all types of IFNs, including IFNα (type I), IFNγ (type II), IFNλ1 and 3 (type III). IFNs canonically induce interferon-stimulated genes (ISGs) to exert their antiviral activities. By profiling a subset of important human ISGs using an overexpression approach, we have identified RTP4 and HPSE as moderate anti-norovirus ISGs, whereas IRF-1, RIG-I (also known as DDX58) and MDA5 (also known as IFIH1) were identified as potent anti-norovirus effectors. Interestingly, type I and III IFNs coordinately induced IRF-1, RIG-I and MDA5; whereas type II IFN predominantly induced IRF-1 to exhibit their anti-norovirus activities. Combination of different IFNs revealed that IFNγ worked cooperatively with type I or type III IFNs to induce ISGs and subsequently inhibit HuNoV replication. Of note, replication of HuNoV did not interfere with antiviral IFN response. In summary, we showed the potent anti-norovirus activities of different types of IFNs and identified the key anti-norovirus effectors. These findings are important for understanding norovirus-host interactions and developing antiviral therapies.

PI3K-Akt-mTOR axis sustains rotavirus infection via the 4E-BP1 mediated autophagy pathway and represents an antiviral target.

Rotavirus infection is a major cause of severe dehydrating diarrhea in infants younger than 5 y old and in particular cases of immunocompromised patients irrespective to the age of the patients. Although vaccines have been developed, antiviral therapy is an important complement that cannot be substituted. Because of the lack of specific approved treatment, it is urgent to facilitate the cascade of further understanding of the infection biology, identification of druggable targets and the final development of effective antiviral therapies. PI3K-Akt-mTOR signaling pathway plays a vital role in regulating the infection course of many viruses. In this study, we have dissected the effects of PI3K-Akt-mTOR signaling pathway on rotavirus infection using both conventional cell culture models and a 3D model of human primary intestinal organoids. We found that PI3K-Akt-mTOR signaling is essential in sustaining rotavirus infection. Thus, blocking the key elements of this pathway, including PI3K, mTOR and 4E-BP1, has resulted in potent anti-rotavirus activity. Importantly, a clinically used mTOR inhibitor, rapamycin, potently inhibited both experimental and patient-derived rotavirus strains. This effect involves 4E-BP1 mediated induction of autophagy, which in turn exerts anti-rotavirus effects. These results revealed new insights on rotavirus-host interactions and provided new avenues for antiviral drug development.

Capsaicin-loaded folic acid-conjugated lipid nanoparticles for enhanced therapeutic efficacy in ovarian cancers.

In this study, folic acid-conjugated lipid nanoparticles were successfully prepared to enhance the active targeting of capsaicin (CAP) in ovarian cancers. The particles were nanosized and exhibited a controlled release of drug in the physiological conditions. The folic acid (FA)-conjugated system exhibited a remarkably higher uptake of nanoparticles in the cancer cells compared to that of non-targeted system. The folate-conjugated CAP-loaded lipid nanoparticles (CFLN) upon interacting with cancer cells were internalized via receptor-mediated endocytosis mechanism and resulted in higher concentration in the cancer cells. Consistently, CFLN showed a remarkably higher toxic effect compared to that of non-targeted nanoparticle system. CFLN showed significantly higher cancer cell apoptosis with nearly 39% of cells in apoptosis chamber (early and late) compared to only ∼21% and ∼11% for CAP-loaded lipid nanoparticles (CLN) and CAP. The loading of drug in the lipid nanoparticle system extended the drug retention in the blood circulation and allowed the active targeting to specific cancer cells. The prolonged circulation of drug attributed to the antifouling property of polyethylene glycol molecule in the structure. Overall, study highlights that using targeting moiety could enhance the therapeutic response of nanomedicines in the treatment of solid tumors.

Dual antifungal properties of cationic antimicrobial peptides polybia-MPI: membrane integrity disruption and inhibition of biofilm formation.

With the increasing emergence of resistant fungi, the discovery and development of novel antifungal therapeutics were urgently needed. Compared with conventional antibiotics, the limited propensity of AMPs to induce resistance in pathogens has attracted great interest. In the present study, the antifungal activity and its mechanism-of-action of polybia-MPI, a cationic peptide from the venom of Social wasp Polybia Paulista was investigated. We demonstrated that polybia-MPI could potently inhibit the growth of Candida albicans (C. albicans) and Candida glabrata (C. glabrata). The 50% inhibitory concentrations (IC50) of Polybia-MPI against cancer cells were much higher than the MICs against the tested C. albicans and C. glabrata cells, indicating that polybia-MPI had high selectivity between the fungal and mammalian cells. Our results also indicated that membrane disturbance mechanism was involved in the antifungal activity. Furthermore, polybia-MPI could inhibit the bio film forming of C. glabrata, which was frequently associated with clinically significant biofilm. These results suggest that polybia-MPI has great advantages in the development of antifungal agents.