key: cord-0791217-korx81ss
authors: Bagheri-Hosseinabadi, Zahra; Pirsadeghi, Ali; Rahnama, Amir; Bahrehmand, Fatemeh; Abbasifard, Mitra
title: Is there any relationship between serum zinc levels and angiotensin-converting enzyme 2 gene expression in patients with coronavirus disease 2019?
date: 2021-11-06
journal: Meta Gene
DOI: 10.1016/j.mgene.2021.100991
sha: 0259860a44071f82bd118681b46c6a66c8d176fa
doc_id: 791217
cord_uid: korx81ss

BACKGROUND: The level of angiotensin-converting enzyme 2 (ACE2) expression in different tissues is essential in the sensitivity, symptoms and consequences of COVID-19 infection. It seems that zinc is involved in the structure of the ACE2 enzyme has been identified; nonetheless, the relationship between ACE2 expression and zinc serum levels in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients is still unclear. This study aimed to evaluate the expression of ACE2 in peripheral blood-derived immune cells of COVID-19 patients and its relationship with serum zinc levels. METHODS: Thirty healthy subjects and thirty patients with COVID-19 were enrolled in this study. The COVID-19 infection was confirmed by positive real-time polymerase chain reaction (RT-PCR) and radiological data. Peripheral blood samples were taken from healthy subjects and COVID-19 patients. Whole blood samples were also used to measure ACE2 gene expression by RT-PCR technique. The correlation matrix evaluated the relationship between ACE2 expression, serum zinc levels, and other related variables. RESULTS: The outcomes showed no considerable alteration in serum zinc levels between patients and the control group. Likewise, the ACE2 gene expression results showed a significant decrease in this receptor's expression in COVID-19 patients compared with the healthy subjects. A significant positive correlation was observed between serum zinc level and ACE2 gene expression in patients with COVID-19. CONCLUSION: The immune system seems to reduce the mRNA expression of the ACE2 in the peripheral blood leukocytes following SARS-CoV-2 infection. Moreover, zinc deficiency can make patients more susceptible to SARS-CoV-2 infection.

Coronaviruses (CoV) are considered a large family of viruses responsible for various disorders, from the common cold to the more severe respiratory disorders and even death [1] . The new coronavirus, SARS-CoV-2, is emerging not previously reported in humans [2] . The virus is transmitted between people through droplets or direct contact, and the average incubation period is reported as 6.4 days [3] . During a preliminary period in an infected patient, the virus is replicated in the upper respiratory tract. After this period, the lower respiratory tract cells' infection leads to infiltration of immune cells, the formation of an inflammatory milieu and lesions, as well as lung tissue injury [4] . Evidence showed that the main symptoms of COVID-19 patients are fever, sore throat, dry cough, runny nose, headaches, body aches, fatigue, diarrhea and vomiting, as well as discoloration of fingers or toes and skin rash [5] . It has been reported that severe symptoms including pneumonia and shortness of breath were observed in 14% of patients; however, only in 5% of patients, the condition was severe, which is associated with acute respiratory distress syndrome (ARDS), infectious shock, multi-organ failure, and even death [6] . The ACE2 gene encodes the ACE2, an enzyme located on the outer surface of epithelial cells in the lungs, blood vessels, heart, kidneys, and intestines [7, 8] . ACE2 is a receptor for the SARS-CoV-2 to enter the target cells [9] . The spike (S) surface glycoprotein of SARS-CoV-2 can bind to the ACE2 receptor, allowing the virus to enter the target cells, particularly epithelial type1 and 2 in the respiratory tract tissue [10] . Therefore, this receptor plays a crucial role in tissue infection susceptibility, clinical symptoms, and consequence of SARS-CoV-2 infection [11] . Previous studies reported a positive and significant association between ACE2 expression and SARS-CoV-2 infection in vitro [12, 13] .

Moreover, the ACE2 enzyme, as a metalloprotein, can chelate and retain zinc due to its reduced zinc levels favor the interaction of the angiotensin-converting motif [14] . Zinc is considered one of the essential trace elements in the human body. Since there is no zinc storage system in the body, zinc's daily consumption is essential to maintain a stable body condition [15] . Studies have shown that zinc is essential for the optimum of several enzyme activities, cellular processes, including growth, DNA synthesis, and RNA transcription [16] . Fluctuations in the intracellular concentration of zinc are essential to inhibit virus transcription [17] . In this context, studies have shown that the binding and elongation pattern of RNA-dependent RNA polymerase (RdRP, J o u r n a l P r e -p r o o f RDR) or RNA replicase in SARS is inhibited by zinc [18] . In vitro studies also showed that high concentrations of zinc and the addition of complexes that induce zinc entry into the cell could inhibit the proliferation of RNA-positive viruses [18] . Protease activity was also inhibited by zinc in rhinovirus and poliovirus [19, 20] .

Further details on the effect of zinc on SARS-CoV-2 infection are currently mysterious. Based on the latest studies, zinc's role in the ACE2 structure has been identified; nonetheless, the relationship between ACE2 expression and serum zinc level in COVID-19 patients is still unclear. Therefore, this study aimed to measure the serum level of zinc and gene expression of ACE2 in peripheral blood-derived immune cells of COVID-19 patients compared to healthy subjects, as well as the association between gene expression of ACE2 with zinc levels and other related variables.

In this investigation, thirty patients with COVID-19 and thirty healthy age and sex-matched subjects were enrolled. Inclusion criteria for COVID-19 patients were hospitalization in Ali Ibn Abitaleb Hospital (Rafsanjan, Iran), positive RT-PCR test and radiological data, and inflammation signs. Subjects with other viruses' infection, respiratory system-related disorders, immune-based diseases such as autoimmunity, allergies, cancer, and immunocompromised patients were excluded from the study. The subjects' demographic and clinical data, including age, sex, hospitalization duration, type of medication before and during the infection, disease outcome (discharge, death), underlying diseases, duration symptom onset, the severity of patients' symptoms (CT scan result, chest x-ray data), and vital signs of patients, were recorded.

Additionally, laboratory parameters such as leukocyte count, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), platelet (PLT) count were also collected. Informed consent has been received from each patient and control group based on the Declaration of Helsinki (DoH). Furthermore, this study has been approved by the Ethics Committee of Rafsanjan University of Medical Sciences, Rafsanjan, Iran (IR.RUMS.REC.1399.025).

Blood samples were collected from both patients and healthy subjects and divided into two fractions for serum separation, cell blood count, and RNA extraction. After collection, the sample was transferred to the laboratory, and isolated serums were kept at -20 ° C until further experiments. The blood indexes, CRP, and ESR levels were measured using appropriate methods and laboratory instruments. In addition, serum zinc level was measured with a Hitachi 912 biochemistry autoanalyzer (Roche, Germany), and absorption was measured at 530-570 nm based on a calorimetric method.

Total RNA was extracted from fresh blood samples using an RNA extraction kit (Karmania pars gene, Iran). Extracted RNAs were evaluated for purity and integrity by spectrophotometry (260/280 nm) and agarose gel electrophoresis. Next, cDNAs were synthesized using a cDNA synthesis kit (Karmania pars gene, Iran) with a 15µL cDNA master mix and a 5µL RNA template (1ng to 5 µg). The cDNA synthesize procedure was performed regarding the one-step protocol suggested by the mentioned manufacturer: 42-50°C for 30 min, followed by 90°C for 5 min for reverse transcriptase (RT) enzyme inactivation, and finally, the microtubes were cooled on the ice for 2 minutes, and 1/5 ratio (1 for cDNA and 5 for total volume) for PCR amplification was used for next step. Also, specific forward and reverse primers (concentration of the primers: 0.5 µM), 2X qPCRBIO SYGreen Mix Hi-ROX (PCRBiosystem, England), and nuclease-free water were prepared for RT-PCR and amplification using a Rotor-Gene Q 2plex System (Qiagen) regarding the recommended program: 1 cycle of 95 °C for 2 minutes; 40 cycles of 95 °C for 5 seconds, and 60-65°C (annealing) for 20 to 30 seconds. Additionally, the melting curve step was used later by 10 seconds at 95 °C and then 10 seconds each at 0.2 °C enhancements between 62 and 95 °C. The designed sequence primers of the studied genes are shown in Table   1 . The RT-PCR was completed in triplicate, and U6 was employed as the reference gene to standardize the acquired signals. Furthermore, the relative expression of the amplified products was correspondingly calculated by the 2 -ΔΔCt formula.

GraphPad Prism 8 (GraphPad Software, San Diego, CA) was employed to perform statistical analysis. The one-sample Kolmogorov-Smirnov (KS) and Shapiro-Wilk tests were used for the variable's normality evaluation. Differences between the groups in the study were also assessed J o u r n a l P r e -p r o o f using independent sample T-test, Mann-Whitney U, and Chi-square statistic tests. Furthermore, the correlation matrix test was used to estimate the association between targeted variables. All data are presented as mean ± SEM. A p-value<0.05 was considered statistically significant.

Thirty patients with COVID-19, including 17 women and 13 men with a mean age of 60±3.39 years and thirty healthy individuals, including 19 women and 11 men with a mean age of 57±1.98 years, enrolled in the study. The statistical analysis results showed no significant difference between the patient and control groups regarding age and sex (P>0.05). The demographic and clinical data of the study groups are shown in Table 2 . The results showed that in the patient group, the expression of the ACE2 gene had a significant decrease compared to the control group (P<0.0001) ( Figure 1 ). Furthermore, serum zinc levels were not significantly different in patients and controls (P=0.06) (Figure 2 ). Statistical analysis and correlation matrix test also showed a positive and significant correlation between serum zinc level and ACE2 gene expression (r=0.44, p=0.02). Also, a significant negative correlation was observed between the number of neutrophils and lymphocytes, which shows that in patients with COVID-19, the severity of the disease increases, and the progression of the infection increases active neutrophils and decreases lymphocytes (r=-0.66, p<0.0001) (Figure 3 ).

This study investigated the expression of the ACE2 gene and its relationship with serum zinc levels. The results of this study showed that there was no significant difference in serum zinc levels between patients with COVID-19 and healthy individuals. However, ACE2 gene expression was significantly reduced in the patient group compared to the control. The correlation results showed a positive and significant relationship between serum zinc levels and ACE2 gene expression.

Recent studies reported that SARS-CoV-2 might infect ACE2 + dendritic cells (DCs), macrophages, and monocytes. However, infection of these immune system cells can also occur in an ACE2-independent pathway through ligation with CD147 and CD209/CD209L [4, 21] . Additionally, pro-inflammatory mediators could be produced by infected macrophages and monocytes, resulting in local and systemic inflammation and cytokine storm, which play a pivotal role in expanding COVID-19-associated disorders, such as ARDS in infected patients with infected patients SARS-CoV-2 [21] . Evidence proposes that ACE2 is regulated by several pathways [23, 24] . In line with this study's results, it has been shown that ACE2 is significantly down-regulated in COVID-19 [25] . Although few studies have been done in this area, there is evidence that zinc can affect virus-induced alterations in B and T cells [26] . It has also been argued that zinc can reduce ACE2 expression [27] . This study showed that despite the lack of significant differences between the patient and control groups in the serum level of zinc, the amount of this trace element in both groups was less than normal, and both groups were zinc deficient. Due to this zinc deficiency, reduced ACE2 expression in patients with COVID-19 may have other unknown mechanisms. Consistent with our data, another study also reported that a large number of patients with COVID-19 had zinc deficiency, developing more complications, and the deficiency was associated with an extended hospital stay and enlarged mortality rates [28] . An experimental investigation reported that zinc with a concentration of 100 µM could decrease recombinant human ACE2 activity in rat lungs [29] . However, the modulation of SARS-CoV-2 infection by administration of high doses of zinc is still hypothetical and needs further study [30] .

Interestingly, zinc as an immune regulator can participate in both the inflammatory and antiinflammatory responses in viral infections. Studies have shown that zinc can increase IFN-α production on the one hand and reduce the pathological inflammation leading to cytokine storm by inhibiting the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling pathway. Zinc can also activate regulatory T cells (Tregs), supporting the homeostasis process [31] . However, the association between zinc levels and ACE2 gene expression in patients' peripheral blood cells with COVID- 19 has not yet been determined.

In contrast with this study's findings, a flow cytometric investigation demonstrated slight to no ACE2 expression by peripheral blood-derived immune cells and platelets while alveolar macrophages can significantly express ACE2 [32] . Furthermore, in vitro investigations have revealed that decreased zinc levels support the interaction of ACE2 with SARS-CoV-2 S protein and elevated zinc levels hinder ACE2 expression, reducing viral interaction, while the statistical analysis in this study showed that there was a positive and significant association between serum level of zinc and ACE2 expression by peripheral blood-derived immune cells in patients with COVID-19 [9, 33] . These discrepancies in outcomes may be due to patients' conditions, treatment protocol, and other unknowns. The finding of an investigation demonstrated that zinc supplementation increased the suppressive impacts of emetine and triclabendazole on the expression of ACE2 in Calu-3 and H322M cells (lung cancer cell lines) [34] . These findings indicated that the ACE2 expression could be modulated by the reactive oxygen species (ROS) and NF-κB signaling in human lung cells, and the combination of triclabendazole or emetine with zinc may have acceptable clinical outcomes for the treatment of patients with COVID-19 by regulating of ACE2 expression [34] .

Regarding the findings of our study conducted on leukocytes of patients with COVID-19, there was a positive and significant association between serum levels of zinc and the expression of ACE2. In this context, studies showed that targeted cell subsets by COVID-19 in host tissues and the regulators of ACE2 expression remain unidentified. It has been revealed that ACE2 might regulate immune response through activation of immunological signaling pathways such as the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) in the SARS-Cov-2 infection, resulting in the activation of helper T cells 1 (Th1 cells), B cells, macrophages and the inhibition of CD8 + T cells and Foxp3 + Tregs [35] . Therefore, in patients under study, factors other than zinc may have played a role in increasing ACE2 expression, as ACE2 expression has been shown to increase significantly 72 hours after SARS-CoV-2 infection, which can occur in both zinc-deficient patients and patients with sufficient zinc levels [35] . Regrettably, due to the invasiveness of the biopsy method from the damaged lung tissue and the dissatisfaction of patients and their families, the measurement of ACE2 tissue expression was limited.

Collectively, this study's results showed that zinc plays an essential role in modulating immune responses in people with COVID-19 and can also play a protective role against viral infections J o u r n a l P r e -p r o o f such as SARS-CoV-2 because serum levels in most studied patients are lower than normal. This zinc deficiency can make patients more susceptible to the infection. On the other hand, gene expression of ACE2 was reduced, although this decrease in mRNA expression was detected in peripheral blood cells and required further studies on the ACE2 protein level in various tissues such as the lung epithelium.

Another decade, another coronavirus

The epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (COVID-19) in China. Zhonghua liu xing bing xue za zhi= Zhonghua liuxingbingxue zazhi

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and corona virus disease-2019 (COVID-19): the epidemic and the challenges

The bio-mission of interleukin-6 in the pathogenesis of COVID-19: A brief look at potential therapeutic tactics. Life sciences

Immunopathological similarities between COVID-19 and influenza: Investigating the consequences of Co-infection

COVID-19 and multi-organ response

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

A novel angiotensinconverting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9

ACE2 receptor polymorphism: Susceptibility to SARS-CoV-2, hypertension, multi-organ failure, and COVID-19 disease outcome

SARS-CoV-2 induces transcriptional signatures in human lung epithelial cells that promote lung fibrosis

Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations

Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor

The S proteins of human coronavirus NL63 and severe acute respiratory syndrome coronavirus bind overlapping regions of ACE2

Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase

Trace elements in human physiology and pathology: zinc and metallothioneins

Clinical, biochemical, and pharmacological role of zinc

The role of zinc in antiviral immunity

Zn2+ inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture

Purification, properties, and mutagenesis of poliovirus 3C protease

Cleavage of small peptides in vitro by human rhinovirus 14 3C protease expressed in Escherichia coli

Contribution of monocytes and macrophages to the local tissue inflammation and cytokine storm in COVID-19: Lessons from SARS and MERS, and potential therapeutic interventions. Life sciences

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly decimates human spleens and lymph nodes

Regulation of ACE2 in cardiac myocytes and fibroblasts

Epigenetic regulation of angiotensin-converting enzyme 2 (ACE2) by SIRT1 under conditions of cell energy stress

COVID-19: the role of excessive cytokine release and potential ACE2 downregulation in promoting hypercoagulable state associated with severe illness

The potential impact of zinc supplementation on COVID-19 pathogenesis

Zinc and respiratory tract infections: Perspectives for COVID-19

COVID-19: Poor outcomes in patients with zinc deficiency

Concentration-dependent effects of zinc on angiotensin-converting enzyme-2 activity (1067.4). The FASEB journal

The effects of coronavirus on human nasal ciliated respiratory epithelium

Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target

High expression of angiotensin-converting enzyme-2 (ACE2) on tissue macrophages that may be targeted by virus SARS-CoV-2 in COVID-19 patients

Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety of human tissues. Infectious diseases of poverty

Zinc supplementation augments the suppressive effects of repurposed NF-κB inhibitors on ACE2 expression in human lung cell lines

The potential involvement of JAK-STAT signaling pathway in the COVID-19 infection assisted by ACE2

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