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*

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

Coronaviruses are single-stranded RNA viruses that are common in animals. In the past 20 years, there have been three large-scale epidemics of coronaviruses, including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease (COVID). Heart disease is an independent risk factor for severe COVID. At the same time, SARS-CoV-2 infection is often complicated with myocardial injury, which is closely related to poor prognosis. The receptors of SARS coronavirus are angiotensin-converting enzyme 2 (ACE2) and CD209L, among which ACE2 is the main receptor, and ACE2 is abundant in the heart. The receptor of MERS-coronavirus is dipeptide peptidase 4 (DPP4), which is not expressed in myocardial cells, but existed in vascular endothelial cells and blood. These receptors are important factors for the myocardial injury caused by coronavirus infection.
Animals ; COVID-19 ; Angiotensin-Converting Enzyme 2 ; SARS-CoV-2 ; Endothelial Cells ; Peptidyl-Dipeptidase A/genetics*

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

New threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design. Here, we identified a receptor-binding domain (RBD)-binding antibody, XG014, which potently neutralizes β-coronavirus lineage B (β-CoV-B), including SARS-CoV-2, its circulating variants, SARS-CoV and bat SARSr-CoV WIV1. Interestingly, antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibody-dependent SARS-CoV-2 spike (S) protein-mediated cell-cell fusion, suggesting a unique mode of recognition by XG014. Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional "down" conformation, while its family member XG005 directly competes with ACE2 binding and position the RBD "up". Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo. Our findings suggest the potential to develop XG014 as pan-β-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines against β-CoV-B and newly emerging SARS-CoV-2 variants of concern.
Angiotensin-Converting Enzyme 2 ; Antibodies, Neutralizing ; Antibodies, Viral ; COVID-19 ; Epitopes ; Humans ; SARS-CoV-2/genetics* ; Spike Glycoprotein, Coronavirus/genetics*

BACKGROUNDCurrently, there are still divergent opinions about the mechanisms of the impaired neovascularization in diabetic subjects. Due to the remarkable therapeutic effect of angiotensin-converting enzyme inhibititors (ACEIs) on the reduction of blood pressure and the protection of target organs, the clinical application of this kind of drugs is very widespread. However, it is still not clear about the role and related molecular pathway of this kind of drugs in the limbs' postischemic revascularization. It is of major therapeutic importance to resolve these questions. This study aimed to investigate the reasons of the impaired angiogenesis in the hind limbs of rats with diabetic ischemia, the role and related molecular mechanisms of ACEI in postischemic revascularization.

METHODSHind limbs ischemia was induced in diabetic rats by right femoral artery excision. Diabetic rats were randomly allocated to one of the following treatments for 4 weeks: ACEI by perindopril; perindopril in combination with a nitric oxide synthase (NOS) inhibitor; perindopril in combination with bradykinin (BK)-B1 receptor (B1R) antagonist or saline. The differences of angiogenesis, the mRNA and protein expression of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and basic fibroblast (bFGF), constitutive nitric oxide synthase (cNOS) activity and nitric oxide (NO) content were observed after treatment.

RESULTSIn non-ischemic hind limbs, no significant changes in capillary density, or the mRNA and protein expression of eNOS, VEGF and bFGF, or the NO content and the cNOS activity were observed among all groups. On the contrary, in ischemic hind limbs, the capillary density in diabetic rats decreased by 27% when compared with the control rats, so did the mRNA and protein expression of eNOS, VEGF and bFGF, or the NO content and the cNOS activity (P < 0.05). The capillary density was increased by 1.65-fold in the perindopril treatment group in reference to untreated diabetic rats. Moreover, administration of perindopril enhanced the mRNA expression of eNOS, VEGF, and bFGF by 1.45-, 1.44-, and 1.33-fold, increased the protein content of the above indices by 1.55-, 1.30- and 1.50-fold compared with the untreated diabetic rats respectively. Perindopril also increased NO content and cNOS activity to 1.33- and 1.38-fold of that in untreated diabetic rats. The combination of BK-B1R antagonist significantly decreased the above indices (P < 0.05). In contrast, the combination of NOS inhibitor decreased the expression of eNOS and bFGF, the NO content and the cNOS activity, while the expression of VEGF did not change.

CONCLUSIONSDiabetes mellitus reduces the neovascularization, related growth factors expression and activity in the diabetic rat ischemic legs model. Treatment of perindopril improves postischemic revascularization. This effect is mediated, at least in part, by the BK-B1R-related pathway, and the activation of VEGF/eNOS/bFGF signals may be involved in the pro-angiogenic effect.

Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Blood Glucose ; analysis ; Diabetes Mellitus, Experimental ; physiopathology ; Extremities ; blood supply ; Fibroblast Growth Factor 2 ; genetics ; Neovascularization, Physiologic ; drug effects ; Nitric Oxide ; analysis ; Nitric Oxide Synthase Type III ; metabolism ; Perindopril ; pharmacology ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Streptozocin ; Vascular Endothelial Growth Factor A ; genetics

BACKGROUNDExcessive deposition of extracellular matrix (ECM) in the kidney is the hallmark of diabetic nephropathy. Increased matrix synthesis has been well documented but the effects of diabetes on degradative pathways, particularly in the in vivo setting. The renal protective effect of these pathways on matrix accumulation has not been fully elucidated. The present study was undertaken to investigate the activity of matrix metalloproteinase-2 (MMP-2), the expression of MMP-2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) in kidney tissues of diabetic rats, and to explore the degradative pathway of type IV collagen (IV-C) and the renal protective effects of ACE inhibition-benazepril.

METHODSTwenty-four healthy male Wistar rats were divided randomly into normal control group (NC group), untreated diabetes mellitus group (DM group), and diabetes mellitus group treated with benazepril (DL group). The rat model of diabetes mellitus was induced by intraperitoneal injection of streptozocin (60 mg/kg). After the establishment of DM model, benazepril (10 mg.kg(-1).d(-1)) was given to the DL group for 12 weeks, and the same volume of water was given to the other two groups. At the end of 12 weeks, renal function was evaluated with 24-hour urinary protein (Upro), clearance of creatinine (Ccr), and blood urea nitrogen (BUN). MMP-2 activity was determined by gelatin zymography. The levels of MMP-2, TIMP-2 and collagen IV (IV-C) protein in the kidney tissue were assessed by immunohistochemistry. The gene expression of MMP-2 and TIMP-2 was measured by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThe levels of BUN, Upro and Ccr in the DM group were higher than those in the NC group. In the DM group, the mRNA, enzymatic activity and proteins of MMP-2 decreased, but the expressions of IV-C and TIMP-2 increased. All diabetes-associated changes in renal function and MMP/TIMP were attenuated after benazepril treatment with reduced IV-C accumulation.

CONCLUSIONSThe changes of MMP-2 and TIMP-2 expressions in kidney tissues of diabetes rats may contribute to the occurrence and progression of diabetic nephropathy. Benazepril could exert protective effects on diabetic nephropathy, owing to the upregulation of MMP-2 and downregulation of TIMP-2 expressions, which further inhibits the excessive deposition of extracellular matrix in the glomerulus.

Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Benzazepines ; pharmacology ; Blood Glucose ; analysis ; Body Weight ; Collagen Type IV ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; Diabetic Nephropathies ; prevention & control ; Kidney ; drug effects ; metabolism ; Kidney Glomerulus ; pathology ; Male ; Matrix Metalloproteinase 2 ; analysis ; genetics ; Organ Size ; Rats ; Rats, Wistar ; Streptozocin ; Tissue Inhibitor of Metalloproteinase-2 ; analysis ; genetics

OBJECTIVETo investigate the effects of pravastatin, fosinopril and their combination on ventricular remodeling, cardiac function, tumor necrosis factor-alpha (TNF-alpha) mRNA expression, and matrix metalloproteinases (MMPs) activities after myocardial infarction (MI) in rats.

METHODSAcute myocardial infarction (AMI) was established by ligation of the anterior descending coronary artery in male Sprague-Dawly (SD) rats. Twenty-four hours after the procedure, the 48 surviving rats were grouped randomly as AMI control, fosinopril (10 mg.kg(-1).d(-1)), pravastatin (20 mg.kg(-1).d(-1)) and a combined use of the 2 drugs. Sham-operated group (n = 8) was taken randomly as non-infarction control. Six weeks after treatment with the drugs by gastric gavage, heart function and left ventricular remodeling were assessed. Left ventricular weight (LVW)/body weight (BW) ratio was determined. The relative expression of myocardium TNF-alpha mRNA was assessed by reverse transcription-polymerase chain reaction. Left ventricular myocardium MMPs activities were assessed by Zymography.

RESULTSThere were no significant differences among the four AMI groups in infarction size (P > 0.05). In comparison with the AMI group, left ventricular end-diastolic pressure, left ventricular end-diastolic diameter, LVW/BW all decreased significantly (P < 0.05 - 0.01); while dp/dtmax, dp/dtmin, fractional shortening (FS) and ejection fraction (EF) increased significantly in all three drug-treated groups (P < 0.05 - 0.01); increments of FS, LVEF and dp/dtmax were more evident in the combination group than either the fosinopril or pravastatin group (P < 0.05). The levels of TNF-alpha mRNA in AMI rats treated with fosinopril, pravastatin and their combination reduced 29%, 26% and 33%, respectively (P < 0.01); MMP-2 activity reduced 25%, 30% and 35%, respectively (P < 0.01); MMP-9 activity reduced 20%, 18% and 24%, respectively (P < 0.01). There were no significant differences in other variables among the 3 treatment groups (P > 0.05).

CONCLUSIONPravastatin, fosinopril and their combination showed favorable effects on left ventricular remodeling after AMI in rats and demonstrated improved cardiac function. The combined treatment group yielded better results in the context of improving left ventricular systolic function. These effects could be relevant to the attenuation of increased MMP-2 and MMP-9 activities and left ventricular expression of TNF-alpha.

Angiotensin-Converting Enzyme Inhibitors ; therapeutic use ; Animals ; Drug Therapy, Combination ; Fosinopril ; administration & dosage ; therapeutic use ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Myocardial Infarction ; drug therapy ; pathology ; physiopathology ; Pravastatin ; administration & dosage ; therapeutic use ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; genetics ; Ventricular Remodeling ; drug effects