Objective This study aims to investigate the effect of berberine (Ber) on foam cell formation induced by oxidized low-density lipoprotein (ox-LDL) in macrophages and to explore the mechanism's association with the ACE2-Ang(1-7)-Mas axis. Methods They were randomly divided into blank group, model group (RAW264.7 cells induced with 60 μg/mL ox-LDL), and berberine group (the model treated with berberine interventions at 2.5, 5, and 10 μmol/L concentrations). Lipid accumulation within the cells was assessed by Oil Red O staining, and the content of lipid droplets in each group was quantitatively analyzed by enzymatic method. The content of total cholesterol (TC) and free cholesterol (FC) in foam cells were detected by enzymatic method. The levels of oxidative stress factors (malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH)), inflammatory factors such as tumor necrosis factor α(TNF-α), and nitric oxide (NO) were measured using corresponding relevant reagent kits. The mRNA and protein expressions of ACE2 and Mas were evaluated through quantitative real-time PCR and Western blot analysis, respectively. The levels of AngII and Ang(1-7) were detected by ELISA. Results Compared with the model group, the berberine groups exhibited reduced lipid droplet accumulation and a dose-dependent decrease in intracellular lipid content. Berberine significantly lowered TC and FC levels in foam cells and reduced the CE/TC ratio. The levels of the oxidative factor MDA were significantly reduced, while the levels of the antioxidant factors SOD and GSH were markedly increased. Inflammatory factors TNF-α and NO were significantly decreased. The expression of the ACE2-Ang(1-7)-Mas signaling pathway was significantly activated, and the effect was more pronounced in the Ber group with high-concentration compared to the group with low-concentration, demonstrating a dose-dependent response. Conclusion Berberine can inhibit macrophage foam cell formation, potentially through upregulation of the ACE2-Ang(1-7)-Mas signaling pathway, thereby contributing to the alleviation of atherosclerosis.
Berberine/pharmacology* ; Foam Cells/cytology* ; Animals ; Signal Transduction/drug effects* ; Mice ; Angiotensin-Converting Enzyme 2 ; Angiotensin I/genetics* ; Peptidyl-Dipeptidase A/genetics* ; Peptide Fragments/genetics* ; Receptors, G-Protein-Coupled/genetics* ; RAW 264.7 Cells ; Proto-Oncogene Proteins/genetics* ; Proto-Oncogene Mas ; Lipoproteins, LDL/pharmacology* ; Nitric Oxide/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

Neutralizing antibodies have been designed to specifically target and bind to the receptor binding domain (RBD) of spike (S) protein to block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus from attaching to angiotensin converting enzyme 2 (ACE2). This study reports a distinctive nanobody, designated as VHH21, that directly catalyzes the S-trimer into an irreversible transition state through postfusion conformational changes. Derived from camels immunized with multiple antigens, a set of nanobodies with high affinity for the S1 protein displays abilities to neutralize pseudovirion infections with a broad resistance to variants of concern of SARS-CoV-2, including SARS-CoV and BatRaTG13. Importantly, a super-resolution screening and analysis platform based on visual fluorescence probes was designed and applied to monitor single proteins and protein subunits. A spontaneously occurring dimeric form of VHH21 was obtained to rapidly destroy the S-trimer. Structural analysis via cryogenic electron microscopy revealed that VHH21 targets specific conserved epitopes on the S protein, distinct from the ACE2 binding site on the RBD, which destabilizes the fusion process. This research highlights the potential of VHH21 as an abzyme-like nanobody (nanoabzyme) possessing broad-spectrum binding capabilities and highly effective anti-viral properties and offers a promising strategy for combating coronavirus outbreaks.
Single-Domain Antibodies/immunology* ; Spike Glycoprotein, Coronavirus/metabolism* ; SARS-CoV-2/immunology* ; Animals ; Humans ; Antibodies, Neutralizing/immunology* ; Camelus ; COVID-19/immunology* ; Antibodies, Viral/immunology* ; Angiotensin-Converting Enzyme 2

The spike (S) protein plays a crucial role in the entry of SARS-CoV-2 into host cells. The S protein contains two subunits, S1 and S2. The receptor-binding domain (RBD) of the S1 subunit binds to the receptor angiotensin-converting enzyme 2 (ACE2) to enter the host cells. Therefore, degrading S1 is one of the feasible strategies to inhibit SARS-CoV-2 infection. The purpose of this study is to develop a degradation tool targeting S1. First, we constructed a HEK 293 cell line stably expressing S1 by using a three-plasmid lentivirus system. The overexpression of the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) in this cell line promoted the ubiquitination of S1 and accelerated its proteasomal degradation. Further research showed the polyubiquitination of S1 catalyzed by MUL1 mainly occurred via the addition of K48-linked chains. Moreover, the specific peptide LCB1, which targets and recognizes S1, was combined with MUL1 to create the chimeric E3 ubiquitin ligase LCB1-MUL1. In comparison to MUL1, this chimeric enzyme demonstrated improved catalytic efficiency, resulting in a reduction of S1's half-life from 12 h to 9 h. In summary, this study elucidated the mechanism by which MUL1 promotes the ubiquitination modification of S1 and facilitates its degradation through the proteasome, and preliminarily validated the effectiveness of targeted degradation of S1 by chimeric enzyme LCB1-MUL1.
Ubiquitin-Protein Ligases/genetics* ; Humans ; HEK293 Cells ; Ubiquitination ; Spike Glycoprotein, Coronavirus/genetics* ; SARS-CoV-2/metabolism* ; Recombinant Fusion Proteins/metabolism* ; Proteasome Endopeptidase Complex/genetics* ; COVID-19/metabolism* ; Angiotensin-Converting Enzyme 2/genetics*

The goal of this study was to investigate the regulatory effect of angiotensin converting enzyme 2 (ACE2) on cellular inflammation caused by avian infectious bronchitis virus (IBV) and the underlying mechanism of such effect. Vero and DF-1 cells were used as test target to be exposed to recombinant IBV virus (IBV-3ab-Luc). Four different groups were tested: the control group, the infection group[IBV-3ab-Luc, MOI (multiplicity of infection)=1], the ACE2 overexpression group[IBV-3ab Luc+pcDNA3.1(+)-ACE2], and the ACE2-depleted group (IBV-3ab-Luc+siRNA-ACE2). After the cells in the infection group started to show cytopathic indicators, the overall protein and RNA in cell of each group were extracted. real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression level of the IBV nucleoprotein (IBV-N), glycoprotein 130 (gp130) and cellular interleukin-6 (IL-6). Enzyme linked immunosorbent assay (ELISA) was used to determine the level of IL-6 in cell supernatant. Western blotting was performed to determine the level of ACE2 phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). We found that ACE2 was successfully overexpressed and depleted in both Vero and DF-1 cells. Secondly, cytopathic indicators were observed in infected Vero cells including rounding, detaching, clumping, and formation of syncytia. These indicators were alleviated in ACE2 overexpression group but exacerbated when ACE2 was depleted. Thirdly, in the infection group, capering with the control group, the expression level of IBV-N, gp130, IL-6 mRNA and increased significantly (P < 0.05), the IL-6 level was significant or extremely significant elevated in cell supernatant (P < 0.05 or P < 0.01); the expression of ACE2 decreased significantly (P < 0.05); protein phosphorylation level of JAK2 and STAT3 increased significantly (P < 0.05). Fourthly, comparing with the infected group, the level of IBV-N mRNA expression in the ACE2 overexpression group had no notable change (P > 0.05), but the expression of gp130 mRNA, IL-6 level and expression of mRNA were elevated (P < 0.05) and the protein phosphorylation level of JAK2 and STAT3 decreased significantly (P < 0.05). In the ACE2-depleted group, there was no notable change in IBV-N (P > 0.05), but the IL-6 level and expression of mRNA increased significantly (P < 0.05) and the phosphorylation level of JAK2 and STAT3 protein decreased slightly (P > 0.05). The results demonstrated for the first time that ACE2 did not affect the replication of IBV in DF-1 cell, but it did contribute to the prevention of the activation of the IL-6/JAK2/STAT3 signaling pathway, resulting in an alleviation of IBV-induced cellular inflammation in Vero and DF-1 cells.
Animals ; Chlorocebus aethiops ; Humans ; Interleukin-6/genetics* ; Janus Kinase 2/pharmacology* ; Infectious bronchitis virus/metabolism* ; STAT3 Transcription Factor/metabolism* ; Angiotensin-Converting Enzyme 2/pharmacology* ; Cytokine Receptor gp130/metabolism* ; Vero Cells ; Signal Transduction ; Inflammation ; RNA, Messenger

Objective To explore the effects and mechanisms of a traditional Chinese medicine (TCM) prescription, "Fang-gan Decoction" (FGD), in protecting against SARS-CoV-2 spike protein-induced lung and intestinal injuries in vitro and in vivo.Methods Female BALB/c mice and three cell lines pretreated with FGD were stimulated with recombinant SARS-CoV-2 spike protein (spike protein). Hematoxylin-eosin (HE) staining and pathologic scoring of tissues, cell permeability and viability, and angiotensin-converting enzyme 2 (ACE2) expression in the lung and colon were detected. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of inflammatory factors in serum and cell supernatant. The expression of NF-κB p65, p-NF-κB p65, p-IκBα, p-Smad2/3, TGF-β1, Caspase3, and Bcl-2 was evaluated by Western blotting.Results FGD protected against the damage to the lung and colon caused by the spike protein in vivo and in vitro according to the pathologic score and cell permeability and viability (P<0.05). FGD up-regulated ACE2 expression, which was reduced by the spike protein in the lung and colon, significantly improved the deregulation of inflammatory markers caused by the spike protein, and regulated the activity of TGF-β/Smads and NF-κB signaling.Conclusion Traditional Chinese medicine has a protective effect on lung and intestinal tissue injury stimulated by the spike protein through possible regulatory functions of the NF-κB and TGF-β1/Smad pathways with tissue type specificity.
Mice ; Animals ; Female ; Humans ; NF-kappa B/metabolism* ; Spike Glycoprotein, Coronavirus/pharmacology* ; Transforming Growth Factor beta1/metabolism* ; Angiotensin-Converting Enzyme 2/pharmacology* ; COVID-19 ; SARS-CoV-2/metabolism* ; Lung ; Antineoplastic Agents ; Transforming Growth Factor beta/pharmacology* ; Epithelial Cells/metabolism* ; Colon

OBJECTIVE: To observe the clinical effect of moxibustion with deqi on Alzheimer's disease (AD) rats, and evaluate its effect on β-amyloid (Aβ) transport and enzymatic degradation proteins, to explore its molecular mechanism for improving cognitive function. METHODS: Sixty SPF-grade male SD rats were randomly divided into a blank group (8 rats), a sham-operation group (8 rats) and a model establishment group (44 rats). The rats in the model establishment group were injected with Aβ₁-₄₂ at bilateral ventricles to establish AD model. Among the 38 rats with successful model establishment, 8 rats were randomly selected as the model group, and the remaining rats were treated with mild moxibustion at "Dazhui" (GV 14), once a day, 40 min each time, for 28 days. According to whether deqi appeared and the occurrence time of deqi, the rats were divided into a deqi group (12 rats), a delayed deqi group (10 rats) and a non-deqi group (8 rats). After the intervention, the Morris water maze test was applied to evaluate the cognitive function; the HE staining was applied to observe the brain morphology; the Western blot method was applied to measure the protein expression of Aβ and its receptor mediated transport [low-density lipoprotein receptor-related protein (LRP) 1, receptor for advanced glycation end products (RAGE), apolipoprotein E (ApoE)] and enzymatic degradation [neprilysin (NEP), insulin degrading enzyme (IDE), endothelin converting enzyme (ECE)-1 and angiotensin converting enzyme (ACE) 2]. RESULTS: Compared with the sham-operation group, in the model group, the escape latency was prolonged (P<0.01), and the times of platform crossing and the ratio of platform quadrant to total time were reduced (P<0.01); the brain tissue was seriously damaged; the expression of hippocampal Aβ and RAGE was increased (P<0.01), and the expression of hippocampal LRP1, ApoE, NEP, IDE, ECE-1 and ACE2 was decreased (P<0.01). Compared with the model group, the escape latency was shortened in the deqi group (P<0.05, P<0.01), and the escape latency in the delayed deqi group and the non-deqi group was shortened from Day 2 to Day 5 (P<0.05, P<0.01), and the times of platform crossing and the ratio of platform quadrant to total time were increased in the deqi group and the delayed deqi group (P<0.01, P<0.05); the brain damage in each moxibustion group was reduced, which was smallest in the deqi group, followed by the delayed deqi group and the non-deqi group; the expression of Aβ and RAGE was decreased (P<0.01, P<0.05) and the expression of LRP1 and IDE was increased in each moxibustion group (P<0.01, P<0.05); the expression of ApoE was increased in the deqi group and the delayed deqi group (P<0.01, P<0.05); the expression of NEP was increased in deqi group (P<0.05), and the expression of ECE-1 and ACE2 was increased in the deqi group and the delayed deqi group (P<0.05). Compared with the delayed deqi group and the non-deqi group, the escape latency in the deqi group was shortened from Day 3 to Day 5 (P<0.05), and the times of platform crossing and the ratio of platform quadrant to total time were increased (P<0.05, P<0.01). Compared with the non-deqi group, the expression of Aβ was reduced (P<0.05), the expression of LRP1 and ApoE was increased in the deqi group (P<0.05). The expression of NEP in the deqi group was higher than that in the delayed deqi group and the non-deqi group (P<0.05). CONCLUSION Compared with non-deqi, moxibustion with deqi could promote Aβ transport and degradation, thereby reducing Aβ level in the brain and improving cognitive function for AD rats.
Alzheimer Disease/therapy* ; Amyloid beta-Peptides/genetics* ; Angiotensin-Converting Enzyme 2 ; Animals ; Apolipoproteins E/metabolism* ; Hippocampus/metabolism* ; Male ; Moxibustion ; Rats ; Rats, Sprague-Dawley

Coronavirus disease (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), with strong contagiousness, high susceptibility and long incubation period. cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain of the viral spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). Here, we briefly reviewed the mechanisms underlying the interaction between SARS-CoV-2 and ACE2, and summarized the latest research progress on SARS-CoV-2 neutralizing monoclonal antibodies and nanobodies, so as to better understand the development process and drug research direction of COVID-19. This review may facilitate understanding the development of neutralizing antibody drugs for emerging infectious diseases, especially for COVID-19.
Angiotensin-Converting Enzyme 2 ; Antibodies, Monoclonal ; Antibodies, Neutralizing ; Antibodies, Viral ; COVID-19 ; Humans ; Peptidyl-Dipeptidase A/metabolism* ; Protein Binding ; SARS-CoV-2 ; Single-Domain Antibodies ; Spike Glycoprotein, Coronavirus/metabolism*

BACKGROUND: Since 2019, a novel coronavirus named 2019 novel coronavirus (2019-nCoV) has emerged worldwide. Apart from fever and respiratory complications, acute kidney injury has been observed in a few patients with coronavirus disease 2019. Furthermore, according to recent findings, the virus has been detected in urine. Angiotensin-converting enzyme II (ACE2) has been proposed to serve as the receptor for the entry of 2019-nCoV, which is the same as that for the severe acute respiratory syndrome. This study aimed to investigate the possible cause of kidney damage and the potential route of 2019-nCoV infection in the urinary system. METHODS: We used both published kidney and bladder cell atlas data and new independent kidney single-cell RNA sequencing data generated in-house to evaluate ACE2 gene expression in all cell types in healthy kidneys and bladders. The Pearson correlation coefficients between ACE2 and all other genes were first generated. Then, genes with r values larger than 0.1 and P values smaller than 0.01 were deemed significant co-expression genes with ACE2. RESULTS: Our results showed the enriched expression of ACE2 in all subtypes of proximal tubule (PT) cells of the kidney. ACE2 expression was found in 5.12%, 5.80%, and 14.38% of the proximal convoluted tubule cells, PT cells, and proximal straight tubule cells, respectively, in three published kidney cell atlas datasets. In addition, ACE2 expression was also confirmed in 12.05%, 6.80%, and 10.20% of cells of the proximal convoluted tubule, PT, and proximal straight tubule, respectively, in our own two healthy kidney samples. For the analysis of public data from three bladder samples, ACE2 expression was low but detectable in bladder epithelial cells. Only 0.25% and 1.28% of intermediate cells and umbrella cells, respectively, had ACE2 expression. CONCLUSION This study has provided bioinformatics evidence of the potential route of 2019-nCoV infection in the urinary system.
Angiotensin-Converting Enzyme 2/metabolism* ; COVID-19 ; Gene Expression ; Humans ; Kidney/metabolism* ; SARS-CoV-2 ; Sequence Analysis, RNA ; Single-Cell Analysis ; Urinary Bladder/metabolism*

Angiotensin-Converting Enzyme 2/metabolism* ; COVID-19/virology* ; Disease Susceptibility ; Humans ; SARS-CoV-2/pathogenicity* ; Species Specificity

With the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) all over the world, there is an increasing number of children with such infection. Angiotensin-converting enzyme 2 (ACE2), one of the binding sites for SARS-CoV-2 infection in humans, can bind to viral spike proteins, allowing transmembrane serine protease (TMPRSS2) to activate S-protein to trigger infection and induce the production of various inflammatory factors such as interleukin-1, interferon-l, and tumor necrosis factor. Compared with adults, children tend to have lower expression levels of ACE2 and TMPRSS2, which are presumed to be associated with milder symptoms and fewer cases in children. The article summarizes the research advances in the role of ACE2 during SARS-CoV-2 infection, in order to help understand the pathogenic mechanism of SARS-CoV-2 and provide a reference for better development of drugs and vaccines to prevent and treat coronavirus disease 2019 in children.
Angiotensin-Converting Enzyme 2/metabolism* ; COVID-19 ; Child ; Humans ; Receptors, Virus/metabolism* ; SARS-CoV-2 ; Serine Endopeptidases/metabolism*