Ursodeoxycholic acid reduces ACE-2 activity in COVID-19 patients and Calu- 3 cells

doi:10.21203/rs.3.rs-5317838/v1

Background

Reportedly, ursodeoxycholic acid (UDCA) decreases Angiotensin-converting enzyme 2 (ACE2) activities by inhibiting FXR to prevent SARS-CoV-2 infection.

Methods

Covid-19 patients (n=142, male=72, female=70) from January to May 2023 were divided into UDCA-free (n=53) and UDCA (n=89) groups and treated withnirmatasvir/ritonavir or molnupiravir for 5 days. Patients in the UDCA group were additionally given UDCA for 10 days. ACE2 was detected and clinical outcomes were assessed. Calu-3 cells were infected with the Covid-19 Spike (XBB.1.5) pseudovirusand incubated with or without UDCA.

Results

On day 0 (before treatment), 3, 6, 9 (after anti-viral drug and/or UDCA treatment), ACE2 in serum and plasma in UDCA-free group was ~41 ng/ml (p=0.9962), and ~68ng/ml (p=0.6179); in UDCA group from 40.1±9.6 to 20.8±5.8 ng/ml (p=0.0000), and 68.8±15.6 to 30.2±7.7 ng/ml ( p=0.0000). In UDCA group, ACE2 mRNA in blood cells was from ~100% to 58.5±13.2% (p=0.000) on day 6 and time for fever return to normal shorter (p=0.0001). In Calu-3 cells, UDCA reduced ACE2 protein and mRNA, and blocked Covid-19 pseudovirus infection.

Conclusion

UDCA reduces ACE2 activity in Covid-19 patients and Calu-3 cells, blocks Covid-19 pseudovirus infection in Calu-3 cells and improves the clinical outcomes. UDCA may be a potential drug for prevention and treatment of SARS-CoV-2 infection.

UDCA

ACE2

SARS-CoV-2

Covid-19

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is a single-stranded RNA β-coronavirus. SARS-CoV-2 contains structural proteins, including spike (S), nucleoside (N), membrane (M) and envelope (E) proteins [1]. SARS-CoV-2 variants include Alpha, Beta, Gamma, Delta, and Omicron. Recently, WHO listed BA.2.86 as variants of interest (VOIs)and JN.1 as variants under monitoring (VUMs) [2]. Angiotensin-converting enzyme 2 (ACE2) is an essential receptor in the cell membrane and binds to the spike (S) protein for mediating the entry of SARS-CoV-2 into cells [3]. Brevini et al have reported that farnesoid X receptor (FXR) is a direct regulator of ACE2 at the transcriptional level. As an existing generic drug for the treatment of cholestatic liver disease, ursodeoxycholic acid (UDCA) reduces ACE2 expression by inhibiting FXR in human lung and liver tissues[4]. In a retrospective cohort of liver transplant recipients, UDCA also improved clinical outcomes following SARS-CoV-2 [4]. Based on UDCA inhibition of FXR that enhances epithelial ACE2 expression [5], we have investigated the effect of UDCA on ACE2 activity in Covid-19 patients and human Calu-3 lung adenocarcinoma cells. In the present study, we have found that UDCA reduces ACE2 expression at the transcriptional and translational levels, improves clinical outcomes, and blocks Covid-19 Spike pseudovirus infection.

Ethics approval and consent to participate

The patient part of the study was conformed to the Declaration of Helsinki (1964) and the protocol was approved by the Ethics Committee of Huzhou Central Hospital (Ethics No: 2023001-02). All human subjects and consent provided written informed consent. Clinical trial number: not applicable.

Patients

Covid-19 patients (n=142, 72 males and 70 females) were evaluated for mild, moderate, severe, and critical stages, at the Department of Infectious Diseases of Huzhou Central Hospital, from January to May 2023. The patients were randomly divided into the UDCA-free (n=53) and the UDCA (n=89) groups, with body temperature of 38.6±1.0 and 38.9±0.8ºC (p=0.554) without significant difference (Table 1).

Treatment

Patients in UDCA-free group (n=53) received Nematevir (300 mg)/ritonavir (100 mg) antiviral therapy twice a day for 5 days (n=45, 85.0%) or Monoravir (800 mg) twice a day for 5 days (n=8, 15.0%). Patients in UDCA group (n=89) received nirmatavir (300 mg)/ritonavir (100 mg) twice a day for 5 days (n=78, 87.6%) or molnupiravir (800 mg) twice a day for 5 days (n=11, 12.4%), in addition, UDCA (0.75 g/d) for 10 days.

Detection of ACE2 levels in human blood samples

Sera, plasma, and blood cells were collected from assessed Covid-19 patients on Day 0 (pre-treatment), 3, 6, and 9 (post-treatment) according to the standard protocols. Sandwich ELISA was performed using a 96-well ELISA plate according to the standard protocol. Reagents included the capture antibody, rabbit anti-ACE2 monoclonal antibody (mAb) (Abcam); AEC2 protein (Abcam) as a standard antigen, serum, or plasma as antigens for detection; primary antibody, mouse anti-ACE2 mAb (Abcam); secondary antibody, HRP-labeled goat anti-mouse IgG. A standard curve of ACE2 concentrations was calculated to determine ACE2 concentration in each sample. Western blots with mouse anti-ACE2 mAb (Abcam) as the primary antibody were used to detect ACE2 bands in plasma.

Cell culture

Calu-3 epithelial cells (human lung adenocarcinoma cells) (ATCC HTB-55) were cultured in MEM medium containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO₂. Cells were spread and cultured in antibiotic-free medium in 10-cm plates for 24 h until the cells reached ~80% conference, followed by incubation of UDCA at 0, 20, 100, 200 μM for 48 h. Cell lysates were analyzed by Western blots for ACE2 expression levels.

Detection of ACE2 mRNA levels by real-time qRT‑PCR

The Total RNA Extraction Kit (Solarbio Life Science, Beijing) was used to extract the total RNA from whole blood cells of Covid-19 patients in UDCA-free and UDCA groups or Calu-3 cells. PCR primers were as follows: ACE2, forward 5'-CCA CTG CTC AAC TAC TTT GAG CC-3' and reverse 5'-CTT ATC CTC ACT TTG ATG CT-3'; GAPDH, forward 5'-GAA GGT GAA GGT CGG AGT C-3' and reverse 5'-GAA GAT GGT GAT GGG ATT TC-3' [6]. The total RNA of each sample was reverse transcribed into the complementary DNA by Perfect Real Time Kit (Takara Bio Inc, Japan). The real-time Quantative PCR (qPCR) was performed with TB Green Permix Ex TagII Kit (Takara) in ABI QuantStudio 5 (Thermofisher). The target gene expression was calculated using 2^-^DD^CT method.

Pseudovirus infection and immunofluorescence staining

Calu-3 cells were placed on 1.4-cm coverslips in the well of a 24-well plate and incubated for 24 h. After washing with PBS, cells were incubated in 0.2 mL medium without or with 0.2 μL of Covid-19 Spike (XBB.1.5) protein pseudovirus (Yeasen Biotechnology, Shanghai, Co, Ltd) for 6 h. After removing the infection medium, cells were incubated in 0.2 mL medium without or with UDCA (200 µM) for 24 h. Cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) and permeabilized with 0.1% Triton X-100 in PBS. After blocking with 1% bovine serum albumin (BSA) in PBS, cellswere incubated with rabbit anti-ACE2 mAb (Abcam) and then incubated with Alexa Fluor 488 F(ab')-goat anti-rabbit IgG (H+L) (Invitrogen). The stained cells were dried in air and then mounted with Vectashield mounting medium for fluorescent H-100 containing 4'-6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc., Burlingame, CA, USA). The cell morphology and immunofluorescence were observed and captured using Leica fluorescence microscope (Wetzlar, Germany).

Statistical analysis

ANOVA was applied to define differences between treatment groups, followed by post-hoc multiple comparison tests to define specific group differences (Statpages). Student t-test was applied to assess the relationship between different variables (GraphPad, Prism). A value of p<0.05 is considered significant, and p<0.01 very significant.

UDCA reduced ACE2 levels in blood samples of Covid-19 patients

Our primary goal was to observe whether UDCA affects ACE2 activity in Covid-19 patients. By detection using Sandwich ELISA, ACE-2 levels in serum and plasma of Covid-19 patients were significant differences between UDCA-free and UDCA groups. On day 0 (before treatment), day 3, 6 and 9 (after taking antiviral drugs and/or UDCA), ACE-2 concentrations in serum and plasma in UDCA-free group kept ~41ng/mL (p=0.9962) and ~68 ng/mL (p=0.6179), but in UDCA group from 40.1±9.6 to 20.8±5.8 ng/mL (p=0.0000) and from 68.8±15.6 to 30.2±7.7 ng/mL (p=0.0000) (Figure 1, A, a, b; Table 2). Comparison of ACE2 concentrations in serum and in plasma between UDCA-free and UDCA groups resulted the significant differences on day 3, 6 and 9 (Figure 1, A, c; Table 2). ACE2 mRNA levels in blood cells of Covid-19 patients were detected by real-time qRT-PCR. On day 0 (before drug treatment), day 3 and day 6 (after taking antiviral drugs and/or UDCA), the levels of ACE-2 mRNA in UDCA-free group were ~100.0% (p=0.4133) without significant differences, in UDCA group 100.0±16.3%, 78.2±17.7%, and 58.5±13.2% (p=0.000) with significant differences (Figure 1, B; Table 2). The data suggest that (1) ACE2 concentrations in circulation system keep in a constant statue in Covid-19 patients; (2) ACE2 enzyme activity is significantly reduced in serum and plasma after treatment with UDCA; (3) ACE-2 levels in plasma are higher than that in serum. Therefore, UDCA reduces ACE2 activity at transcriptional and translational levels in Covid-19 patients.

UDCA affected ACE2 in plasma of Covid-19 patients

Western blotting showed ACE2 in plasma (Figure 1, C). In UDCA-free group the ACE2 bands on day 0, 3, 6 and 9 were similar in thickness (Figure 1, C, 1, 3, 5, 7) but in UDCA group the band on day 0, 3, 6 and 9 became thinner and thinner (Figure 1, C, 2, 4, 6,8).The results suggest that UDCA reduces ACE2 levels in the plasma of Covid-19 patients.

UDCA shortened period of recovery from fever in Covid-19 patients

The effect of UDCA on clinical outcomes was also observed. In UDCA-free (n=53) and UDCA groups (n=89), the time of recovery from fever to normal body temperature was 2.79±1.81 days and 1.43±0.86 days (p=0.0001) with a significant difference (Table 1). The time to respiratory improvement was 3.27±1.55 days and 3.24±1.60 days (p=0.9131) with no significant difference (Table 1), indicating that UDCA improves the clinical outcome of fever returning to normal but does not affect respiratory symptoms in Covid-19 patients.

UDCA reduced the ACE2 levels in Calu-3 cells

Calu-3 cells were incubated with 0, 25, 100 or and 200 μM of UDCA for 48 h. Western blots showed that ACE2 expression levels in cell lysates were 100.0±4.0%, 54.3±2.1%, 53.9±4.5%, and 17.2±9.3% (p=0.0000) with significant difference (Figure 2A; Table 2). ACE2 mRNAlevels detected by real-time qRT-PCR were 100.7±4.7%, 74.6±8.2%, 65.6±5.0% and 39.3±7.2% (p=0.0000) with significant differences (Figure 2, B; Table 2). The results suggest that UDCA reduces ACE2 at the transcription and translational levels in Calu-3 cells.

UDCA decreased ACE2 immunofluorescence signal and UDCA blocked the infection of Covid-19 pseudovirus

To observe the effect of UDCA on ACE2 expression in lung cells, Calu-3 cells were incubated without or with UDCA (200 μM) for 48 h. Immunofluorescence staining showed ACE2 signal (red) in Calu-3 cells (Figure 2, C, a). After incubation with 200 μM of UDCA, the red ACE2 signal was slighter (Figure 2, C, b). Covid-19-Spike (XBB.1.5) protein pseudovirus uses a retroviral vector, in which the envelope protein gene is replaced by the SARS-CoV-2 spike protein gene and the green fluorescent protein (GFP) gene. Covid-19 spike fluorescent signal (green) was appeared in pseudovirus-infected calu-3 cells (Figure 2, C, c) and weaker in pseudovirus-infected calu-3 cells co-incubated with UDCA (200 μM) for 48 h (Figure 2, C, d). In uninfected Calu-3 cells, ACE2 signaling (red) was present, but no Covid-19 spike signal was observed (Figure 2, D, a). After infection with the Covid-19-Spike (XBB.1.5) protein pseudovirus, the spike signal (green) was clearly observed (Figure 2, D, b). After incubation with UDCA, both the ACE2 signal (red) and the spike signal (green) were much slighter (Figure 2, D, c). The data suggest that (1) UDCA reduces the ACE2 fluorescence signal in Calu-3 cells; (2) Covid-19-Spike (XBB.1.5) protein pseudovirus successfully infects Calu-3 cells with a green fluorescence signal; (3) UDCA decreases ACE2 expression that may block pseudovirus infection.

Recently, mixed results have been reported about UDCA improving clinical outcomes in chronic liver disease patients with SARS-CoV-2 infection [7-9]. In tissue culture experiments, UDCA reduces ACE2 expression in different types of cells [4, 10-12].In this study, Covid-19 patients received antiviral therapy with nirmatrelvir/ritonavir or molnupiravir for 5 days and/or UDCA for 10 days. By ELISA measurement, the concentrations of ACE2 in serum and plasma ranged from 10 to 100 ng/ml ACE2 levels in plasma were higher than that in serum. After Covid-19 patients started taking UDCA, ACE2 concentrations in serum and plasma were significantly decreased from day 3 to day 9. ACE2 mRNA levels detected by real-time qRT-PCR are also reduced after UDCA treatment. After taking UDCA, it takes less time for Covid-19 patients to return to normalfrom fever. We have cultured human lung adenocarcinoma Calu-3 epithelial cells with different concentrations of UDCA, resulting that UDCA effectually decreases ACE2 protein and mRNA levels. We have also observed that UDCA reduces ACE2 expression and blocks infection of Covid-19 spike (XBB.1.5) protein pseudovirus infection in Calu-3 cells.Our study provides positive data of UDCA in the prevention and treatment of SARS-CoV-2 infection. Further experiments are needed to understand the molecular mechanism by which UDCA reduces ACE2 activity.

We have compared ACE2 activity in Covid-19 patients treated with or without UDCA and found that UDCA reduces ACE2 levels in serum and plasma, and ACE2 mRNA in blood cells, which may lead to a shorter time to return to normal body temperature. UDCA can also reduce ACE2 levels and block Covid-3 spike pseudovirus infection in Calu-3 cells. These findings may provide evidence for the use of UDCA in prevention and treatment of SARS-CoV-2 infection.

Ethics approval and consent to participate

The patient part of the study was conformed to the Declaration of Helsinki (1964) and the protocol was approved by the Ethics Committee of Huzhou Central Hospital (Ethics No: 2023001-02). All human subjects and consent provided written informed consent.

Clinical Trial

Clinical trial number: not applicable.

Consent for publication

The Author confirms: that the work described has not been published before; that it is not under consideration for publication elsewhere; that its publication has been approved by all co-authors; that its publication has been approved by the responsible authorities at Huzhou Central Hospital where the work is carried out. The author warrants that his/her contribution is original and that he/she has full power to make this consent. The author signs for and accepts responsibility for releasing this material on behalf of any and all co-authors. The copyright transfer covers the exclusive right to reproduce and distribute the article.

Availability of data and materials

Sequence data that support the findings of this study have been deposited in 6935d52ea566782993608c3813ac8d0e ncbi_dataset/data/gene.fna

ed19c16de360d5af79751346c5b50c4c ncbi_dataset/data/data_report.jsonl

16502604615ec502a914a47f34a3465e ncbi_dataset/data/dataset_catalog.json

Competing interests

No conflicts of interest to disclose. All authors declare that they have no conflicts of interest.

Funding

This work was supported by a grant from Zhejiang Province Public Welfare Research Project (Grant number. LGF22H190006) and Huzhou Public Welfare Application Research Project (Grant number. 2021GZ50).

Author Contributions

Z.T.: conceptualization, investigation, data curation, writing original draft, and project administration. J.Z.: methodology, formal analysis, resources. Q.W.: methodology, sample collection, formal analysis, investigation. F.Q.: validation, formal analysis, investigation. L. Z.: experiment operation, data analysis. W.W.: supervision, project administration, and funding acquisition. K.Q.: methodology, formal analysis, supervision, project administration, writing and revising manuscript, and funding acquisition.

Data Availability Statement

The data underlying this study is openly available.

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Table 1 Patient Characteristics

	UDCA-free group	UDCA group	p
Patients n(%)	53(100.0)	89(100.0)
Male	21 (39.6)	51(57.3)
Female	32(60.4)	38(42.7)
Age (y)	51.6±20.7	61.8±16.1	0.0013
Range (y)	15-86	26-91
Covid-19 stage n(%)
Mild	41(77.4)	17(19.1)
Moderate	3(5.7)	16(18.0)
Severe	8(15.1)	45(50.6)
Critical	1(1.9)	11(12.4)
Temperature (°C) before treatment	38.6±1.1	38.9±0.6	0.0372
Range (°C)	36.3-40.0	36.4-40.0
Symptom n(%)
Cough	46(86.8)	77(86.6)
Coughing up sputum	5(9.4)	41(46.1)
Sore throat	35(66.0)	35(40.7)
Chest tightness	9(17.0)	24(27.0)
Muscle aches	34(64.2)	71(79.8)
Loss of taste	0(0.0)	8(9.0)
Basic diseases
Cirrhosis	10(18.9)	44(49.4)
Chronic hepatitis B	32(60.4)	19(21.3
hypertension and/or heart diseases	2(3.8)	13(14.6)
Antiviral therapy n(%)
Nematevir 300 mg/ritonavir 100 mg 2 time/d for 5 d	45(85.0)	78(87.6)
Monoravir 800 mg 2 time/d for 5 d	8(15.0)	11(12.4)
UDCA 0.75 g/d for 10 d n(%)	0(0.0)	89(100.0)
Time for temperature back to normal (d)	2.79±1.81	1.43±0.86	0.0001
Range (d)	1-9	1-6
Time for respiratory improvement (d)	3.27±1.55	3.24±1.60	0.9131
Range (d)	1-7	1-7

Table 2 Measurement of ACE protein and mRNA levels in blood samples of Covid-19 patients by ELISA and real-time qRT-PCR

A. ACE2 concentration in serum and plasma of Covid-19 patients detected by Sandwich ELISA
	Day	0		3		6		9
		ACE2 ng/ml	n	ACE2 ng/ml	n	ACE2 ng/ml	n	ACE2 ng/ml	n	p
Serum	UDCA-free	41.2±11.8	46	40.9±12.3	43	41.0±11.0	37	41.6±12.9	24	0.9962
	UDCA	40.1±9.6	70	27.8±8.4	63	23.1±8.4	40	20.8±5.8	23	0.0000
	p	0.5965		0.0001		0.0001		0.0001
Plasma	UDCA-free	70.6±13.3	31	68.3±12.6	25	66.7±10.1	22	67.0±10.6	21	0.6179
	UDCA	68.8±15.6	51	49.8±16.0	43	38.1±11.9	38	30.2±7.7	34	0.0000
	p	0.6205		0.0001		0.0001		0.0001
Serum	UDCA-free	41.2±11.8	46	40.9±12.3	43	41.0±11.0	37	41.6±12.9	24	0.9962
Plasma	UDCA-free	70.6±13.3	31	68.3±12.6	25	66.7±10.1	22	67.0±10.6	21	0.6179
	p	0.0001		0.0001		0.0001		0.0001
Serum	UDCA	40.1±9.6	70	27.8±8.4	63	23.1±8.4	40	20.8±5.8	23	0.0000
Plasma	UDCA	68.8±15.6	51	49.8±16.0	43	38.1±11.9	38	30.2±7.7	34	0.0000
	p	0.0001		0.0001		0.0001		0.0001
B. ACE2 mRNA levels in blood cells of Covid-19 patients detected by real-time qRT-PCR
Real-time qRT-PCR	Day	1		3		6
ACE mRNA		2-DDCT	n	2-DDCT	n	2-DDCT	n	p
Blood cells	UDCA-free	100.0±12.7%	38	101.9±15.6	33	97.4±12.0%	31	0.4133
Blood cells	UDCA	100.0±16.3%	54	78.2±17.7%	51	58.5±13.2%	49	0.0001
	p	1.0000		0.0001		0.0001
C. ACE2 leves in Calu-3 cells incubated with UDCA detected by Western blots
Western blots	UDCA µM	0	n	25	n	100	n	200	n	p
Cula-3 cell lysates		100.0±4.0%	3	54.3±2.1%	3	53.9±4.5%	3	17.2±9.3%	3	0.0000
D. ACE2 mRNA levels in Calu-3 cells incubated with UDCA detected by real-time qRT-PCR
ACE mRNA	UDCA µM	0	n	25	n	100	n	200	n	p
Cula-3 cell lysates		100.7±4.7%	7	74.6±8.2%	7	65.6±5.0%	7	39.3±7.2%	7	0.0000

Note: All data are presented as Mean±SD; Analyses by ANOVA and student t test.

No competing interests reported.

Ursodeoxycholic acid reduces ACE-2 activity in COVID-19 patients and Calu- 3 cells

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Abstract

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Discussion

Conclusion

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