Osthole exhibits the remedial potential for polycystic ovary syndrome mice through Nrf2-Foxo1-GSH-NF-κB pathway.

Polycystic ovary syndrome (PCOS) is the primary cause of female infertility with a lack of universal therapeutic regimen. Although osthole exhibits numerous pharmacological activities in treating various diseases, its therapeutic effect on PCOS is undiscovered. The present study found that application of osthole improved the symptoms of PCOS mice through preventing ovarian granulosa cells (GCs) production of more estrogen and alleviating the liberation of pro-inflammatory cytokine interleukin (IL)-1ß, IL-6, and tumor necrosis factor alpha. Meanwhile, osthole enhanced ovarian antioxidant capacity and alleviated intracellular reactive oxygen species (ROS) accumulation with a concurrent attenuation for oxidative stress, while intervention of antioxidant enzymic activity and glutathione (GSH) synthesis neutralized the salvation of osthole on GCs secretory disorder and chronic inflammation. Further analysis revealed that osthole restored the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and forkhead box O 1 (Foxo1) whose repression antagonized the amelioration of osthole on the insufficiency of antioxidant capacity and accumulation of ROS. Moreover, Nrf2 served as an intermedium to mediate the regulation of osthole on Foxo1. Additionally, osthole restricted the phosphorylation of IκBα and nuclear factor kappa B (NF-κB) subunit p65 by DHEA and weakened the transcriptional activity of NF-κB, but this effectiveness was abrogated by the obstruction of Nrf2 and Foxo1, whereas adjunction of GSH renewed the redemptive effect of osthole on NF-κB whose activation caused an invalidation of osthole in rescuing the aberration of GCs secretory function and inflammation response. Collectively, osthole might relieve the symptoms of PCOS mice via Nrf2-Foxo1-GSH-NF-κB pathway.

Puerarin Ameliorated PCOS through Preventing Mitochondrial Dysfunction Dependent on the Maintenance of Intracellular Calcium Homeostasis.

Polycystic ovarian syndrome (PCOS) is the major cause of infertility in reproductive women, but no universal drug is feasible. Although puerarin clinically treats cerebrovascular and cardiovascular diseases, its curative effect on PCOS remains elusive. The present study discovered that administration of puerarin restored estrous cycle of PCOS mice and diminished the number of cystic follicles with the concomitant recovery for circulating testosterone, LH and FSH levels, and LH/FSH ratio, indicating the therapeutic role of puerarin in PCOS. KEGG analysis of differential genes between PCOS and control revealed the enrichment in MAPK and calcium signaling pathway. Application of puerarin restricted the phosphorylation of ERK1/2 and JNK, whose activation neutralized the improvement of puerarin on the secretory function and apoptosis of ovarian granulosa cells (GCs). Meanwhile, puerarin alleviated the accumulation of cytosolic Ca2+ through restricting the opening of Ryr and Itpr channels, but this effectiveness was counteracted by the activatory ERK1/2 and JNK. Attenuation of cytosolic Ca2+ counteracted the antagonistic effects of ERK1/2 and JNK activation on puerarin's role in rescuing the calcineurin and Nfatc. Further analysis manifested that Mcu had been authenticated as a direct downstream target of Nfatc to mediate the amelioration of puerarin on mitochondrial Ca2+ uptake. Moreover, puerarin prevented the disorder of ATP content, mitochondrial membrane potential, and mitochondrial permeability transition pore opening through maintaining mitochondrial Ca2+ homeostasis. Collectively, puerarin might ameliorate the symptoms of PCOS mice through preventing mitochondrial dysfunction that is dependent on the maintenance of intracellular Ca2+ homeostasis after inactivation of ERK1/2 and JNK.

Rosa roxburghii Tratt Pomace Crude Extract Inactivates Cronobacter sakazakii Isolated from Powdered Infant Formula.

Cronobacter sakazakii is an important foodborne pathogen in powder infant formula (PIF). The objective of this study was to evaluate the inactivation effect of Rosa roxburghii Tratt pomace crude extract (RRPCE) on C. sakazakii isolated from PIF and to reveal the mechanism of action. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were used to evaluate the inhibitory activity of RRPCE against C. sakazakii. The inhibitory mechanism was revealed from the perspective of effects of RRPCE on intracellular adenosine 5'-triphosphate (ATP), reactive oxygen species (ROS), membrane potential, protein and nucleic acid leakage, and cell morphology of C. sakazakii. The inactivation effects of RRPCE on C. sakazakii in biofilms on stainless steel, tinplate, glass, silica gel, polyethylene terephthalate, and polystyrene to evaluate its potential as a natural disinfectant. The results showed that the MIC and MBC of RRPCE against C. sakazakii were 7.5 and 15 mg/mL, respectively. After treatments with RRPCE, intracellular ATP content decreased significantly while intracellular ROS level increased significantly (p < 0.05). The cell membrane depolarization, large leakage of proteins and nucleic acids, and severely damaged cell morphology also occurred in C. sakazakii treated with RRPCE. In addition, a 20-minute treatment with 2 MIC (15 mg/mL) of RRPCE could inactivate all C. sakazakii (from 6.10 to 6.40 CFU/mL) in biofilms on all six contact surfaces. Our findings suggest that RRPCE is ideal for the inactivation of C. sakazakii and has the potential to be used as a natural disinfectant for the inactivation of PIF packaging materials and containers.

IgM-specific linear epitopes on the E2 protein for serodiagnosis of Chikungunya.

Chikungunya virus (CHIKV) and Dengue virus (DENV) are vector-borne diseases transmitted by Aedes aegypti and Aedes albopictus that pose a significant threat to global public health. Cases of acute Chikungunya fever often present similar clinical symptoms to other vector-borne diseases, such as Dengue fever. In regions where multiple vector-borne diseases coexist, CHIKV is often overlooked or misdiagnosed as Dengue virus, West Nile virus, Zika virus or other viral infections, which delays its prevention and control. However, IgM antibodies directed against the E2 protein of CHIKV have not yet been generalized to clinical settings due to the low sensitivity and high cost in commercial kits. Indirect ELISA with peptides provides an effective supplementary tool for detecting CHIKV IgM antibodies. Our study aims at examining the potential of linear epitopes on the E2 glycoprotein that specifically bind to IgM antibodies as serodiagnostic tool for CHIKV. The sensitivity of the established peptide indirect ELISA method for detecting clinical samples is significantly better than that of commercial kits, realizing a beneficial supplement to the existing IgM antibody assay. It also established the groundwork for comprehending the biological mechanisms of the CHIKV E2 protein and the advancement of innovative epitope peptide vaccines.

Melatonin modulates endometrial decidualization via NOTCH1-NRF2-FOXO1-GSH pathway†.

Melatonin is important for oocyte maturation, fertilization, early embryonic development, and embryo implantation, but less knowledge is available regarding its role in decidualization. The present study found that melatonin did not alter the proliferation of human endometrial stromal cells (ESCs), as well as cell cycle progress, but suppressed stromal differentiation after binding to the melatonin receptor 1B (MTNR1B), which was visualized in decidualizing ESCs. Further analysis evidenced that application of melatonin resulted in the diminishment for NOTCH1 and RBPJ expression. Supplementation of recombinant NOTCH1 protein (rNOTCH1) counteracted the impairment of stromal differentiation conferred by melatonin, while the addition of the NOTCH signaling pathway inhibitor DAPT aggravated the differentiation progress. Meanwhile, melatonin might restrain the expression and transcriptional activity of nuclear factor erythroid 2-related factor 2 (NRF2), whose blockage accelerated the fault of stromal differentiation under the context of melatonin, but this restraint was subsequently ameliorated by rNOTCH1. Forkhead box O 1 (FOXO1) was identified as a downstream target of melatonin in decidualization. Repression of NRF2 antagonized the retrieval of rNOTCH1 due to aberrant FOXO1 expression elicited by melatonin. Moreover, melatonin brought about the occurrence of oxidative stress accompanied by an obvious accumulation of intracellular reactive oxygen species and a significant reduction in glutathione (GSH) content, as well as enzymatic activities of glutathione peroxidase and glutathione reductase, whereas supplementation of rNOTCH1 improved the above-mentioned effects. Nevertheless, this improvement was disrupted by the blockage of NRF2 and FOXO1. Furthermore, addition of GSH rescued the defect of stromal differentiation by melatonin. Collectively, melatonin might impair endometrial decidualization by restraining the differentiation of ESCs dependent on NOTCH1-NRF2-FOXO1-GSH pathway after binding to the MTNR1B receptor.

[Limited internal fixation combined with a hinged external fixator in treatment of peri-elbow bone infection].

Objective: To evaluate the effectiveness of limited internal fixation combined with a hinged external fixator in the treatment of peri-elbow bone infection. Methods: The clinical data of 19 patients with peri-elbow bone infection treated with limited internal fixation combined with a hinged external fixator between May 2018 and May 2021 were retrospectively analyzed. There were 15 males and 4 females with an average age of 44.6 years (range, 28-61 years). There were 13 cases of distal humerus fractures and 6 cases of proximal ulna fractures. All the 19 cases were infected after internal fixation of fracture, and 2 cases were complicated with radial nerve injury. According to Cierny-Mader anatomical classification, 11 cases were type Ⅱ, 6 cases were type Ⅲ, and 2 cases were type Ⅳ. The duration of bone infection was 1-3 years. After primary debridement, the bone defect was (3.04±0.28) cm, and the antibiotic bone cement was implanted into the defect area, and the external fixator was installed; 3 cases were repaired with latissimus dorsi myocutaneous flap, and 2 cases were repaired with lateral brachial fascial flap. Bone defects repair and reconstruction were performed after 6-8 weeks of infection control. The wound healing was observed, and white blood cell (WBC), erythrocyte sedimentation rate (ESR), and C-reaction protein (CRP) were reexamined regularly after operation to evaluate the infection control. X-ray films of the affected limb were taken regularly after operation to observe the bone healing in the defect area. At last follow-up, the flexion and extension range of motion and the total range of motion of the elbow joint were observed and recorded, and compared with those before operation, and the function of the elbow joint was evaluated by Mayo score. Results: All patients were followed up 12-34 months (mean, 26.2 months). The wounds healed in 5 cases after skin flap repair. Two cases of recurrent infection were effectively controlled by debridement again and replacement of antibiotic bone cement. The infection control rate was 89.47% (17/19) in the first stage. Two patients with radial nerve injury had poor muscle strength of the affected limb, and the muscle strength of the affected limb recovered from grade Ⅲ to about grade Ⅳ after rehabilitation exercise. During the follow-up period, there was no complication such as incision ulceration, exudation, bone nonunion, infection recurrence, or infection in the bone harvesting area. Bone healing time ranged from 16 to 37 weeks, with an average of 24.2 weeks. WBC, ESR, CRP, PCT, and elbow flexion, extension, and total range of motions significantly improved at last follow-up ( P<0.05). According to Mayo elbow scoring system, the results were excellent in 14 cases, good in 3 cases, and fair in 2 cases, and the excellent and good rate was 89.47%. Conclusion: Limited internal fixation combined with a hinged external fixator in the treatment of the peri-elbow bone infection can effectively control infection and restore the function of the elbow joint.

Bending mechanics test and parameters calibration of ramie stalks.

Research and development of ramie harvesting equipment is a key link to revitalize ramie industry, problems such as the tendency of stalks to tangle and clog the machine are very problematic, seriously affect the quality and fluency of the harvester. The structure of ramie stalk is complex, and the mechanical properties of each component vary greatly, collision between stalk and machine creates complex stress relationship. By building a finite element model, it is possible to analyze the stress state of the stalk during bending from a microscopic perspective, and to analyze the complex stress-strain situation within the stalk. The purpose of this paper is to establish a standard ramie stalk bending finite element model to provide a theoretical basis for the subsequent kinematics and dynamics. Firstly, material experiments were carried out on ramie straw. The structural and mechanical parameters of the straw components were obtained through measurement and calculation tests, and the force-deformation curves for straw bending were obtained. Bending finite element simulations were carried out on the basis of mechanical tests, and the parameters such as dynamic friction coefficient, wood Poisson's ratio and bast Poisson's ratio were determined by the central combination design. Then established an accurate bending finite element simulation model of ramie stalk, the accuracy of the model was verified at the end. In this paper, the key parameters of the ramie stalk model were calibrated through a combination of material tests and simulations. All parameters of the ramie stalk model were finally obtained, and the bending mechanical properties of the ramie stalk were analysed by applying finite element analysis. This bending mechanics simulation model can be used for kinematic and dynamics simulation analysis of conveying and baling to provide a theoretical basis for the structural design of the harvester. The methods explored here can be applied to other slender straw crops.

TAZ ameliorates the microglia-mediated inflammatory response via the Nrf2-ROS-NF-κB pathway.

Transcriptional co-activator with PDZ-binding motif (TAZ), one of core modules of the Hippo pathway, involves inflammatory cell infiltration in the liver, but little information is available regarding its physiological function in the microglia-mediated inflammatory response. Here we revealed that activation of TAZ prevented microglia production of proinflammatory cytokines, indicating TAZ's importance in anti-inflammation. After translocation into the nucleus, TAZ interacted with transcriptional enhanced associate domain (TEAD) and bound to the promoter of nuclear factor erythroid 2-related factor 2 (Nrf2), whose blockage caused inability of TAZ to improve inflammation, implying that Nrf2 is a direct target of TAZ. Further analysis showed that TAZ induced Nrf2 nuclear translocation to enhance antioxidant capacity with attenuation of oxidative stress and the inflammatory response. Under inflammatory conditions, TAZ impeded mitochondrial dysfunction, as indicated by amelioration of ATP levels, mtDNA copy numbers, and mitochondrial membrane potential with an obvious reduction in mitochondrial superoxide, but this impediment was neutralized by blockage of Nrf2. TAZ hindered opening of the mitochondrial permeability transition pore, restrained release of cytochrome c from mitochondria into the cytosol, and was sufficient to rescue microglia from apoptosis dependent on Nrf2. Nrf2 acted as a downstream target of TAZ to repress NF-κB activation by enhancing antioxidant capacity. Collectively, TAZ might ameliorate the microglia-mediated inflammatory response through the Nrf2-reactive oxygen species (ROS)-nuclear factor κB (NF-κB) pathway.

Yap is essential for uterine decidualization through Rrm2/GSH/ROS pathway in response to Bmp2.

Yap is required for ovarian follicle and early embryo development, but little information is available regarding its physiological significance in decidualization. Here we determine the effects of YAP on decidualization, mitochondrial function, cell apoptosis and DNA damage, and explore its interplay with Bmp2, Rrm2, GSH and ROS. The results exhibited that Yap was abundant in decidual cells and its inactivation impaired the proliferation and differentiation of stromal cells along with the deferral of G1/S phase transition, indicating Yap importance in decidualization. Bmp2 via Alk2 receptor promoted nuclear translocation of Yap where it might interact with Tead and then bind to the promoter of Rrm2 whose activation rescued the faultiness of differentiation program and attenuated oxidative DNA damage caused by Yap impediment. Meanwhile, Yap had an important part in the crosstalk between Bmp2 and Rrm2. Furthermore, inactivation of Yap resulted in an obvious accumulation of intracellular ROS followed by the abnormal GR activity and GSH content dependent on Rrm2. Replenishment of GSH counteracted the regulation of Yap inactivation on stromal differentiation and DNA damage with distinct reduction for intracellular ROS. Additionally, blockage of Yap caused the enhancement of stromal cell apoptosis and brought about mitochondrial dysfunction as indicated by the aberration for ATP level, mtDNA copy number and mitochondrial membrane potential concomitant with the opening of mitochondrial permeability transition pore, but these abnormalities were neutralized by GSH. Administration of mitochondrial antioxidant Mito-TEMPO rescued the fault of stromal differentiation conferred by Yap inactivation. Collectively, Yap was essential for uterine decidualization through Rrm2/GSH/ROS pathway in response to Bmp2.

HB-EGF induces mitochondrial dysfunction via estrogen hypersecretion in granulosa cells dependent on cAMP-PKA-JNK/ERK-Ca2+-FOXO1 pathway.

Polycystic ovarian syndrome (PCOS) is one of the most prevalent endocrinopathies and the leading cause of anovulatory infertility, but its pathogenesis remains elusive. Although HB-EGF is involved in ovarian cancer progression, there is still no clarity about its relevance with PCOS. The present study exhibited that abundant HB-EGF was noted in follicular fluid from PCOS women, where it might induce the granulosa cells (GCs) production of more estrogen via the elevation of CYP19A1 expression after binding to EGFR. Furthermore, HB-EGF transduced intracellular downstream cAMP-PKA signaling to promote the phosphorylation of JNK and ERK whose blockage impeded the induction of HB-EGF on estrogen secretion. Meanwhile, HB-EGF enhanced the accumulation of intracellular Ca2+ whose chelation by BAPTA-AM abrogated the stimulation of HB-EGF on FOXO1 along with an obvious diminishment for estrogen production. cAMP-PKA-JNK/ERK-Ca2+ pathway played an important role in the crosstalk between HB-EGF and FOXO1. Treatment of GCs with HB-EGF resulted in mitochondrial dysfunction as evinced by the reduction of ATP content, mtDNA copy number and mitochondrial membrane potential. Additionally, HB-EGF facilitated the opening of mitochondrial permeability transition pore via targeting BAX and raised the release of cytochrome C from mitochondria into the cytosol to trigger the apoptosis of GCs, but this effectiveness was counteracted by estrogen receptor antagonist. Collectively, HB-EGF might induce mitochondrial dysfunction and GCs apoptosis through advancing estrogen hypersecretion dependent on cAMP-PKA-JNK/ERK-Ca2+-FOXO1 pathway and act as a promising therapeutic target for PCOS.

Effect of Jinzhen granule on two coronaviruses: The novel SARS-CoV-2 and the HCoV-229E and the evidences for their mechanisms of action.

BACKGROUND: Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus 229E (HCoV-229E) pose a huge threat to human public health, no specific treatment is available. Jinzhen granule (JZ) is a traditional eight ingredients-Chinese medicine with prominent efficacy for treating viral-induced diseases. However, little is known about the antiviral effect and mechanism of JZ against SARS-CoV-2 and HCoV-229E. PURPOSE: This study aimed to reveal the antiviral effects of JZ against SARS-CoV-2 and HCoV-229E, and to further explore the underlying mechanisms regulating the host immune response. METHODS: The chromatographic separation of JZ was performed using a Shimadzu analytical high-performance liquid chromatograph with UV detection and Alltech ELSD 2000ES. We conducted cytopathic effect (CPE) and plaque reduction assays to evaluate the antiviral effect of JZ. A lethal human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 was established to determine the protective effect of JZ on mortality and lung virus titers. Real-time quantitative PCR assays were used to analyze the expression of proinflammatory cytokines in vitro and in vivo. Western blotting was further performed to determine the activities on regulating the nuclear factor kappa B (NF-κB)/MAPK pathway. Finally, mitochondrial membrane potential assays, flow cytometry analysis and western blotting were used to assess the anti-apoptotic potency toward HCoV-229E infection. RESULTS: The results showed that 13 chemical components were identified and five peaks were determined and quantitated (gallic acid 1.97 mg/g, baicalin 20.69 mg/g, glycyrrhizic acid 4.92 mg/g, hyodeoxycholic acid 4.86 mg/g, cholic acid 4.07 mg/g). We found that JZ exerted inhibitory potency against SARS-CoV-2 and HCoV-229E in vitro by using CPE and plaque reduction assays, and it was further found that JZ protected mice infected by SARS-CoV-2 from death and inhibited lung virus titers. JZ also significantly decreased the induction of inflammatory cytokines (IL-1α, IL-6, CCL-5 and MIP-1ß), similar to the observed in vitro effect. Moreover, JZ suppressed the release of inflammatory cytokines in vitro and it decreased the protein expression of p-p38 MAPK, p-JNK, p-NF-κB p65 and p-IκBα induced by HCoV-229E and increased the expression of IκBα. Notably, JZ significantly protected HCoV-229E-infected Huh-7 cells from mitochondrial damage and decreased apoptotic cells. The activation of the mitochondria-mediated apoptotic pathway was inhibited by JZ, as shown by the reduced expression of cleaved caspase-9, caspase-3 and p-PARP. CONCLUSIONS: In conclusion, JZ (gallic acid 1.97 mg/g, baicalin 20.69 mg/g, glycyrrhizic acid 4.92 mg/g, hyodeoxycholic acid 4.86 mg/g, cholic acid 4.07 mg/g) exhibited antiviral activities against SARS-CoV-2 and HCoV-229E by regulating the NF-κB/MAPK pathway and the mitochondria-mediated apoptotic pathway. These findings demonstrated the efficacy of JZ against CoVs and suggested JZ treatment as a novel clinical therapeutic strategy for COVID-19.

Genistein exhibits therapeutic potential for PCOS mice via the ER-Nrf2-Foxo1-ROS pathway.

Polycystic ovarian syndrome (PCOS) is a complex endocrinopathy in women of reproductive age and the main cause of female infertility, but there is no universal drug for PCOS therapy. As a predominant dietary isoflavone present in soybeans, genistein (GEN) possesses estrogenic and antioxidative properties, but limited information is available regarding its therapeutic potential and underlying molecular mechanism in PCOS. In this study, we found that GEN might restore the estrous cycle of PCOS mice and ameliorate the elevation of circulating T, AMH and LH levels as well as LH/FSH ratios along with reduced cystic follicles, indicating the importance of GEN in PCOS therapy. Meanwhile, GEN improved the ovarian secretion function of PCOS mice and attenuated oxidative damage of the ovary through enhancing its antioxidant capability dependent on ER. Supplementation of GEN improved the defect of the ATP level and mitochondrial membrane potential, indicating the significance of GEN in preventing mitochondrial dysfunction. Further analysis demonstrated that GEN via ER heightened the expression of Nrf2 and Foxo1 whose blockage antagonized the defence of GEN on the secretory and mitochondrial functions of ovarian granulosa cells followed by the limited antioxidant capability and increased intracellular ROS level. Moreover, nuclear translocation and transcriptional activity of Nrf2 presented a notable enhancement after exposure to GEN. Addition of the Nrf2 inhibitor ML385 hampered the GEN induction of Foxo1. Nrf2 might directly bind to the antioxidant response element of the Foxo1 promoter region. Collectively, GEN might exhibit therapeutic potential for PCOS mice via the ER-Nrf2-Foxo1-ROS pathway.

Potent prophylactic and therapeutic efficacy of recombinant human ACE2-Fc against SARS-CoV-2 infection in vivo.

The current COVID-19 pandemic, caused by SARS-CoV-2, poses a serious public health threat. Effective therapeutic and prophylactic treatments are urgently needed. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, which binds to the receptor binding domain (RBD) of SARS-CoV-2 spike protein. Here, we developed recombinant human ACE2-Fc fusion protein (hACE2-Fc) and a hACE2-Fc mutant with reduced catalytic activity. hACE2-Fc and the hACE2-Fc mutant both efficiently blocked entry of SARS-CoV-2, SARS-CoV, and HCoV-NL63 into hACE2-expressing cells and inhibited SARS-CoV-2 S protein-mediated cell-cell fusion. hACE2-Fc also neutralized various SARS-CoV-2 strains with enhanced infectivity including D614G and V367F mutations, as well as the emerging SARS-CoV-2 variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.1 (Kappa), and B.1.617.2 (Delta), demonstrating its potent and broad-spectrum antiviral effects. In addition, hACE2-Fc proteins protected HBE from SARS-CoV-2 infection. Unlike RBD-targeting neutralizing antibodies, hACE2-Fc treatment did not induce the development of escape mutants. Furthermore, both prophylactic and therapeutic hACE2-Fc treatments effectively protected mice from SARS-CoV-2 infection, as determined by reduced viral replication, weight loss, histological changes, and inflammation in the lungs. The protection provided by hACE2 showed obvious dose-dependent efficacy in vivo. Pharmacokinetic data indicated that hACE2-Fc has a relative long half-life in vivo compared to soluble ACE2, which makes it an excellent candidate for prophylaxis and therapy for COVID-19 as well as for SARS-CoV and HCoV-NL63 infections.

Mapping and role of T cell response in SARS-CoV-2-infected mice.

Virus-specific T cells play essential roles in protection against multiple virus infections, including SARS-CoV and MERS-CoV. While SARS-CoV-2-specific T cells have been identified in COVID-19 patients, their role in the protection of SARS-CoV-2-infected mice is not established. Here, using mice sensitized for infection with SARS-CoV-2 by transduction with an adenovirus expressing the human receptor (Ad5-hACE2), we identified SARS-CoV-2-specific T cell epitopes recognized by CD4+ and CD8+ T cells in BALB/c and C57BL/6 mice. Virus-specific T cells were polyfunctional and were able to lyse target cells in vivo. Further, type I interferon pathway was proved to be critical for generating optimal antiviral T cell responses after SARS-CoV-2 infection. T cell vaccination alone partially protected SARS-CoV-2-infected mice from severe disease. In addition, the results demonstrated cross-reactive T cell responses between SARS-CoV and SARS-CoV-2, but not MERS-CoV, in mice. Understanding the role of the T cell response will guide immunopathogenesis studies of COVID-19 and vaccine design and validation.

Phillyrin (KD-1) exerts anti-viral and anti-inflammatory activities against novel coronavirus (SARS-CoV-2) and human coronavirus 229E (HCoV-229E) by suppressing the nuclear factor kappa B (NF-κB) signaling pathway.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has extensively and rapidly spread in the world, causing an outbreak of acute infectious pneumonia. However, no specific antiviral drugs or vaccines can be used. Phillyrin (KD-1), a representative ingredient of Forsythia suspensa, possesses anti-inflammatory, anti-oxidant, and antiviral activities. However, little is known about the antiviral abilities and mechanism of KD-1 against SARS-CoV-2 and human coronavirus 229E (HCoV-229E). PURPOSE: The study was designed to investigate the antiviral and anti-inflammatory activities of KD-1 against the novel SARS-CoV-2 and HCoV-229E and its potential effect in regulating host immune response in vitro. METHODS: The antiviral activities of KD-1 against SARS-CoV-2 and HCoV-229E were assessed in Vero E6 cells using cytopathic effect and plaque-reduction assay. Proinflammatory cytokine expression levels upon infection with SARS-CoV-2 and HCoV-229E infection in Huh-7 cells were measured by real-time quantitative PCR assays. Western blot assay was used to determine the protein expression of nuclear factor kappa B (NF-κB) p65, p-NF-κB p65, IκBα, and p-IκBα in Huh-7 cells, which are the key targets of the NF-κB pathway. RESULTS: KD-1 could significantly inhibit SARS-CoV-2 and HCoV-229E replication in vitro. KD-1 could also markedly reduce the production of proinflammatory cytokines (TNF-α, IL-6, IL-1ß, MCP-1, and IP-10) at the mRNA levels. Moreover, KD-1 could significantly reduce the protein expression of p-NF-κB p65, NF-κB p65, and p-IκBα, while increasing the expression of IκBα in Huh-7 cells. CONCLUSIONS: KD-1 could significantly inhibit virus proliferation in vitro, the up-regulated expression of proinflammatory cytokines induced by SARS-CoV-2 and HCoV-229E by regulating the activity of the NF-кB signaling pathway. Our findings indicated that KD-1 protected against virus attack and can thus be used as a novel strategy for controlling the coronavirus disease 2019.

Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment.

COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.

Kinetics of viral load and antibody response in relation to COVID-19 severity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus 2019 (COVID-19) pneumonia. Little is known about the kinetics, tissue distribution, cross-reactivity, and neutralization antibody response in patients with COVID-19. Two groups of patients with RT-PCR-confirmed COVID-19 were enrolled in this study: 12 severely ill patients in intensive care units who needed mechanical ventilation and 11 mildly ill patients in isolation wards. Serial clinical samples were collected for laboratory detection. Results showed that most of the severely ill patients had viral shedding in a variety of tissues for 20-40 days after onset of disease (8/12, 66.7%), while the majority of mildly ill patients had viral shedding restricted to the respiratory tract and had no detectable virus RNA 10 days after onset (9/11, 81.8%). Mildly ill patients showed significantly lower IgM response compared with that of the severe group. IgG responses were detected in most patients in both the severe and mild groups at 9 days after onset, and remained at a high level throughout the study. Antibodies cross-reactive to SARS-CoV and SARS-CoV-2 were detected in patients with COVID-19 but not in patients with MERS. High levels of neutralizing antibodies were induced after about 10 days after onset in both severely and mildly ill patients which were higher in the severe group. SARS-CoV-2 pseudotype neutralization test and focus reduction neutralization test with authentic virus showed consistent results. Sera from patients with COVID-19 inhibited SARS-CoV-2 entry. Sera from convalescent patients with SARS or Middle East respiratory syndrome (MERS) did not. Anti-SARS-CoV-2 S and N IgG levels exhibited a moderate correlation with neutralization titers in patients' plasma. This study improves our understanding of immune response in humans after SARS-CoV-2 infection.

Liu Shen capsule shows antiviral and anti-inflammatory abilities against novel coronavirus SARS-CoV-2 via suppression of NF-κB signaling pathway.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread worldwide through person-to-person contact, causing a public health emergency of international concern. At present, there is no specific antiviral treatment recommended for SARS-CoV-2 infection. Liu Shen capsule (LS), a traditional Chinese medicine, has been proven to have a wide spectrum of pharmacological properties, such as anti-inflammatory, antiviral and immunomodulatory activities. However, little is known about the antiviral effect of LS against SARS-CoV-2. Herein, the study was designed to investigate the antiviral activity of SARS-CoV-2 and its potential effect in regulating the host's immune response. The inhibitory effect of LS against SARS-CoV-2 replication in Vero E6 cells was evaluated by using the cytopathic effect (CPE) and plaque reduction assay. The number of virions of SARS-CoV-2 was observed under transmission electron microscope after treatment with LS. Proinflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays. The results showed that LS could significantly inhibit SARS-CoV-2 replication in Vero E6 cells, and reduce the number of virus particles and it could markedly reduce pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-8, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Moreover, the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blot and it was found that LS could inhibit the expression of p-NF-κB p65, p-IκBα and p-p38 MAPK, while increasing the expression of IκBα. These findings indicate that LS could inhibit SARS-CoV-2 virus infection via downregulating the expression of inflammatory cytokines induced virus and regulating the activity of NF-κB/MAPK signaling pathway in vitro, making its promising candidate treatment for controlling COVID-19 disease.