The Effect of Azithromycin Plus Zinc Sulfate on ACE2 Expression through I κ B α of Human Respiratory Cells in SARS-CoV-2: In Vitro Study

The Effect of Azithromycin Plus Zinc Sulfate on ACE2 Expression through I κ B α of Human Respiratory Cells in SARS-CoV-2: Vitro Study. Abstract: Large-scale efforts have been persistently undertaken for medical prophylaxis and treatment of COVID-19 disasters worldwide. A variety of novel viral spike protein-targeted vaccines have been extensively distributed for global inoculation based on accelerated approval. With concerns of emerging spike protein mutations, we revisited the early but inconclusive clinical interest in the repurposed combination of azithromycin (AZT) and zinc supplements with safety advantages. The aim of this study is to provide in vitro proof of concept for I κ B α associated rapid and synergistic suppression of angiotensin-converting enzymes 2 (ACE2) following combination treatments with AZT plus zinc sulfate in two human airway cells with ACE2 expression, Calu-3 and H322M, representative cells of the human upper and lower airway origin respectively. Clinical timing of AZT combined with zinc is indicated based on suppression of the key cellular entry molecule, ACE2, of SARS-CoV-2.


Introduction
Cell surface angiotensin-converting enzymes 2 (ACE2) of the respiratory tract is a wellestablished critical entry of SARS-CoV-2 into infected cells [1][2][3]. ACE2 mRNA expression was shown to be reduced from tracheobronchial to bronchioloalveolar regions [4]. ACE2 expression was upregulated following viral infection [5,6], interferon exposure [5,6], and smoking [5,6]. It has been postulated that hyperactivation of the transcription factor nuclear factor-κB (NF-κB) following ACE2-mediated viral entry, most likely in nonimmune cells, including lung epithelial cells, resulted in cytokine release syndrome [7]. Human type II pneumocytes (AT2) are one of the primary targets for SARS-CoV-2 infection [6]. It was shown in an induced pluripotent stem cell-derived AT2 model that NF-κB signaling was rapidly and persistently upregulated upon SARS-CoV-2 infection [8]. Delayed interferon (IFN) activation was also observed in the same model [8]. Increased blood type I IFN-α levels were reported 7 days after infection and indicated better clinical outcomes [9]. IFN-α, known to activate NF-κB pathway [10,11], also showed in vitro antiviral activity against COVID 2021, 1 264 SARS-CoV-2 [12]. However, IFN-α increased ACE2 expression in primary nasal epithelial cells from healthy nasal mucosa and a human bronchial cell line [13]. Therefore, interaction between ACE2 and NF-κB is worth explored and targeted for ACE2 suppression. With emerging threats of mutations located on the viral spike protein [14], blocking ACE2 for viral entry has become one of the major ways with prophylactic and early therapeutic attempts to prevent unpredictable inflammation induced by SARS-CoV-2 infection [15,16].
Among a variety of repurposed combinations for COVID-19, regimens containing azithromycin (AZT) [17][18][19][20][21][22][23][24] or zinc supplements [25] and in combinations [26,27] have drawn clinical attention since early outbreak. There was a trend toward better clinical outcomes in retrospective analysis for hydroxychloroquine (HCQ) plus AZT. A pilot observation study of 80 PCR-documented hospitalized patients with relatively mild disease treated with combination of 10-day HCQ and 5-day AZT reported high rate of clinical recovery as well as rapid and viral load clearance [18]. The largest retrospective analysis of 3737 PCR-documented patients treated with the same combination earlier also suggested better clinical outcomes and a faster viral load reduction [19]. Another similar retrospective study with 1061 PCR-documented patients also showed favorable clinical outcomes and a high rate of viral load clearance [23]. Only in one large retrospective cohort with 1438 lab-confirmed patients, in-hospital mortality was not reduced by HCQ with/without AZT [20]. However, clinical benefits of the HCQ/AZT combination were not shown in two representative prospective trials. In a multicenter, randomized, open-label controlled trial involving 667 suspected and confirmed hospitalized patients with mild-to-moderate COVID-19, 7-day HCQ with/without AZT did not improved clinical outcomes [17]. In another randomized, open-labeled trial for 447 hospitalized patients with severe suspected and confirmed COVID-19, AZT in addition to HCQ did not further improve clinical outcomes [24]. With regard to zinc supplement, a retrospective observation between HCQ/AZT with/without zinc sulfate for PCR-documented patients reported favorable clinical outcomes with addition of zinc sulfate [26]. In a retrospective case series, zinc combined with AZT and low-dose HCQ reduced hospitalization rate in PCR-documented risk-stratified community patients [27]. Besides, large-scale randomized trials did not reveal hydroxychloroquine's clinical benefits of in patients with COVID-19 [28,29]. Possibly due to inconclusive clinical roles and cardiac concerns [17,20,24], AZT monotherapy was ever explored in outpatient setting. A randomized, open-label controlled trial involving a larger proportion of suspected patients did not show clinical improvements after three-day outpatient AZT treatment [22]. A single high-dose outpatient AZT did not provide clinical benefits for lab-documented patients in a randomized, placebo-controlled trial [30]. Accordingly, the feasible clinical timing and combination choice with AZT remain unelucidated.
Zinc is a trace element supplement with clinical benefits in respiratory tract infections [38]. Zinc deficiency prevalence of 26% was ever reported in a case-control study of adults aged 50 years or older visiting an Ohio outpatient clinic between 2014 and 2017 [39]. Patients with COVID-19 had significantly lower serum zinc levels than normal controls [40]. More complications are developed in COVID-19 patients with zinc deficiency [40]. The prophylactic and therapeutic roles of zinc supplementation are currently under investigation [41,42]. Prevention of viral entry has been postulated to be a potential mechanism for zinc antiviral actions [38]. Increased NF-κB DNA binding activity and IκBα mRNA expression were reported in lungs from a septic mouse model with zinc deficiency [43].
Based on hypothesis of IκBα-mediated ACE2 suppression for viral entry to human airway cells, the aim of this study was to explore in vitro if synergistic ACE2 suppression played a role in the underlying mechanism of the two potential IκBα modulators, AZT, a zinc ionophore [26], and zinc sulfate. Importantly, it was expected to provide first laboratory support for clinical timing of the repurposed AZT plus zinc early in the outbreak for COVID-19 with less clinical safety concerns of IκBα modulation.

Drug Treatments
In order to screen the potential and dose of Zn and AZT for rapid ACE2 mRNA suppression, H322M was seeded at a density of 5 × 10 5 cells per 6-well plate incubated for 20 h at 37 • C in 5% CO 2 . The culture medium was removed and replaced with fresh medium in the presence of 24 h serial doses of 18.75, 37.5, 75, 150, and 300 µM Zn and 24 h serial doses of 50, 25, 12.5, 6.25 and 3.125 µM AZT respectively.

RNA Isolation
Total RNA was isolated from each treatment and control group using the PureLink TM RNA mini kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The RNA concentration and quality were assessed using the Quibit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA samples were stored at −80 • C.

Reverse Transcription (RT)
RT reactions were carried out using the superscript III first strand synthesis system (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized starting from 2 µg of purified total RNA. The reactions in a final volume of 20 µL contained 1x Buffer, dithiothreitol (DTT), deoxy-ribonucleoside triphosphates (dNTPs), superscript III RT, and 500 ng oligo(dT). Samples were incubated at 65 • C for 5 min and 50 • C for 60 min, and then the RT enzyme was inactivated by heating to 70 • C for 15 min. cDNA samples were stored at −20 • C.

Quantitative PCR (qPCR)
qPCR was carried out with KAPA SYBR ® FAST qPCR Master Mix (Wilmington, MA, USA) in a final volume of 20 µL, with 0.3 µM forward and reverse primer and 1 µL of cDNA. Fluorescent detection was performed using the ABI 7500 fast System (Thermo Fisher Scientific, Waltham, MA, USA) with the following thermal cycling conditions: initial polymerase activation at 95 • C for 3 min, followed by 40 cycles of denaturation at 95 • C for 3 s and annealing/extension at 60 • C for 30 s. After amplification, dissociation (melting) curve analysis was performed to analyze the product melting temperature. Each sample was amplified in triplicate wells. Negative (no template) controls were included in each assay. The results were analyzed using ABI 7500 Fast System Software. The threshold cycle (Ct) at which the amount of amplified target reached a fixed threshold was determined. Relative expression was calculated using the 2 −∆∆Cq method. The results were analyzed and are shown as the fold change relative to each control group. Primers of our targets of interest, ACE2, IKBα, MUC1 were listed in Table 1. Table 1 also included primers of internal control, RPLP0, for Calu-3 and GAPDH for H322M.

Western Blot
Cells were scraped with lysis buffer (1% Triton X-100, 20 mM Tris pH 7.4, 150 mM NaCl, and protease inhibitors) on an ice tray, and cell lysates were subjected to western blot analysis. Protein samples were first separated by SDS-PAGE and then transferred to a PVDF membrane. Primary antibodies were applied to detect specific protein expression, followed by incubation with appropriate HRP-conjugated secondary antibodies. Protein signals were developed using an enhanced chemiluminescence reagent (Biomate, Taipei, Taiwan) and detected by a BIO-RAD ChemiDoc TM MP imaging system (BIO-RAD, Hercules, CA, USA). Western blots were carried out with ACE-2 antibody (Bioss, Woburn, MA, USA), IKBα antibody (Santa Cruz, Dallas, TX, USA), and PARP antibody (Cell Signaling, Danvers, MA, USA); α-tubulin antibody (Novus, Littleton, CO, USA) was used as a loading control.

Statistical Analysis
For statistical analysis, the mean and standard errors were calculated by using GraphPad Prism software version 9 (GraphPad Software Inc., San Diego, CA, USA). Student's t-tests were used to determine significant differences between two experimental conditions. p < 0.05 was considered to be significant for all experiments. Data were presented as mean + SEM.

Discussion
Published clinical repurposing combinations containing AZT, zinc or in combinations are categorized into retrospective analysis and prospective clinical trials. One of major differences between the two categories is that patients in retrospective analysis are labdocumented COVID-19, but a small proportion of only clinically suspected COVID-19 patients were enrolled in prospective trials. Interestingly, clinical benefits of repurposed combinations shown in retrospective analysis were usually not reached in prospective trials. Due to concerns of cardiac events in HCQ component of combinations, this study is Total RNA was collected for MUC1 quantitation by real-time qRT-PCR assays. The data were normalized to GAPDH/RPLP0 expression for H322M/Calu-3 cells respectively and are presented as the mean ± SEM (n = 3). (* p = 0.01 to 0.05, ** p = 0.001 to 0.01, and **** p = < 0.0001). Compared to the control, MUC1 expression was significantly increased following 300 µM Zn treatment alone (H322M, p < 0.0001; Calu-3, p = 0.0013) and to a lesser degree combined with AZT in H322M and Calu-3 cells. In H322M, MUC1 expression was synergistically increased following 50 µM AZT treatments combined with 300 µM Zn compared to AZT alone. In Calu-3 cells, MUC1 expression was synergistically increased following 25 and 50 µM AZT treatments combined with 300 µM Zn compared to AZT alone.

Discussion
Published clinical repurposing combinations containing AZT, zinc or in combinations are categorized into retrospective analysis and prospective clinical trials. One of major differences between the two categories is that patients in retrospective analysis are lab-documented COVID-19, but a small proportion of only clinically suspected COVID-19 patients were enrolled in prospective trials. Interestingly, clinical benefits of repurposed combinations shown in retrospective analysis were usually not reached in prospective trials. Due to concerns of cardiac events in HCQ component of combinations, this study is the first to present in vitro evidence that HCQ-free repurposed combination of AZT plus Zn rapidly and significantly suppresses endogenous ACE2 expression and increases MUC1 expression in Calu-3 and H322M cells. A published computed model of the AZT-Zinc ion omplex demonstrated its potential against the replication and assembly of SARS-CoV-2 particles (doi:10.2174/1874091X02014010033). The Calu-3 cells generated from human proximal bronchial adenocarcinoma [47] are characterized by differentiated, functional human airway epithelial cells [48]. These cells were also proposed to be a suitable model for the human nasal mucosa [49]. Bronchoalveolar lavage analyses from COVID-19 patients disclosed aberrant macrophage and T cell responses [50] as well as bronchoalveolar immune hyperactivation [51]. Our drug repurposing for the critical bronchoalveolar involvement of COVID-19 was investigated with H322M, a bronchioalveolar cell line, in addition to Calu-3 for proximal airway involvement.
Symptoms, including fever, dyspnea and hypoxia, were rapidly improved following high-dose zinc rescue with different preparations in a consecutive COVID-19 case series [25]. A randomized placebo-controlled trial for high-dose intravenous zinc was initiated as adjunctive therapy in SARS-CoV-2-positive critically ill patients [52]. Although the targeted enrollment was not reached, the pilot report showed only minimal infusion site irritation in a small proportion of patients in treatment group [53]. Therefore, it is supposed clinically safe to supplement repurposed AZT with high-dose zinc. Interestingly, high-dose Zn alone in this study showed mild-to-moderate ACE2 suppression in Calu-3 and H322M.
Certain NF-κB dimeric transcription factors and their activities are tightly repressed by three inhibitors, IκBα, IκBβ, and IκBε, through the formation of stable IκB-NF-κB complexes. Zinc is supposed to be an IκBα modulator with antiviral activities including viral entry prevention. It was demonstrated that rapid degradation of free IκBα is critical for NF-κB activation [54]. 50 µM AZT induced significant in vitro antirhinoviral activities in normal primary bronchial epithelial cells [33] and those cells from children with cystic fibrosis [34]. Lower in vitro anti-rhinoviral levels of AZT were also reported for bronchial epithelial cells from patients with chronic obstructive lung disease [35]. In this study, IκBα protein expressions were increased in Calu-3 and H322M following 24 h treatment of high-dose AZT of 50 µM plus Zn. Progressively ACE2 suppression ensued from 24 h to 48 h treatments of the two cells with highdose AZT plus Zn, indicative of sequential regulatory interaction between ACE2 and IκBα. The different patterns of IκBα mRNA changes in Calu-3 and H322M suggested endogenous cell-type specific regulatory effects of AZT and Zn in combinations. The concomitant increased IkBα mRNA/protein in Calu-3 indicated increased protein expression following upregulated mRNA transcription by combination treatments. The inverse changes of IkBα mRNA/protein in H322M supposed to be associated with decreased IkBα degradation following combination treatments.
Hypoxia is a common threat in moderate-to-severe COVID-19. SARS -CoV-2 infection increased lung MUC1 expression and resulted in hypoxia in a mouse model [46]. In order to explore optimal clinical timing of the repurposed combination, this study revealed that 24 h high-dose Zn alone and combined with AZT increased MUC1 mRNA expression of Calu-3 and H322M, especially high-dose Zn. Both ACE2 suppression and increased MUC1 mRNA expression were consistently demonstrated in the two human airway cells, indicating the prophylactic and early therapeutic potential of AZT and Zn repurposing combination for COVID-19. However, further preclinical studies and clinical trials must be performed for validation before clinical decision making.
In vitro cell line exploration using two representative human airway cells is the limitation of this study. If we can verify in animal experiments and compare with actual patient samples, the results will increase clinical application value.

Conclusions
This study demonstrated in vitro proof of concept for IκBα associated rapid and synergistic suppression of the key viral entry molecule, ACE2, following combination treatments of clinically repurposed AZT plus zinc early in COVID-19 outbreak. Taking regulatory potential of AZT plus Zn on MUC1 of human airway cells, such clinical repurposing could be considered early in COVID-19 disease course. Institutional Review Board Statement: Ethical review and approval were waived for this study, due to in vitro cell line study only without biohazard material.

Informed Consent Statement: Not applicable.
Data Availability Statement: Any further information will be available from the corresponding authors on request.