Ursodeoxycholic Acid Reduces Angiotensin‐Converting Enzyme 2 Activity and Suppresses Cytokine Storm Syndrome in Patients With Coronavirus Disease 2019

FIGURE 1. ACE2 activity was decreased in the blood of COVID‐19 patients treated with UDCA. (A) Sandwich ELISA detection of ACE2 concentrations in serum (a) and plasma (b), and comparative analysis of ACE2 levels between serum and plasma over time (c), in UDCA‐free and UDCA groups on Day 0 (before treatment), Day 3, Day 6, and Day 9 (after antiviral and/or UDCA treatment). Individual data points, means, and SD are shown. Serum: UDCA‐free (n = 46, 43, 37, 24) and UDCA (n = 70, 63, 40, 23) at Days 0, 3, 6, 9. Plasma: UDCA‐free (n = 31, 25, 22, 21) and UDCA (n = 51, 43, 38, 34) at Days 0, 3, 6, 9. ANOVA with post hoc tests and/or Student's t‐test: *p < 0.05, **p < 0.01. (B) Real‐time qRT‐PCR detection of ACE2 mRNA levels in blood cells of COVID‐19 patients in UDCA‐free and UDCA groups on Days 0, 3, and 6. Data are presented as mean ± SD, with individual values (UDCA‐free: n = 38, 33, 31; UDCA: n = 54, 51, 49). **p < 0.01. (C) Western blot analysis of ACE2 protein in plasma. Representative bands from UDCA‐free (Lanes 1, 3, 5, 7) and UDCA (Lanes 2, 4, 6, 8) groups at Days 0, 3, 6, and 9 are shown.

FIGURE 2. UDCA reduces ACE2 expression and blocks COVID‐19 spike pseudovirus infection in Calu‐3 cells. (A) Calu‐3 cells are incubated with 0, 25, 100, or 200 µM of UDCA for 48 h. (a) Representative Western blot showing ACE2 protein expression in whole‐cell lysates with β‐actin as a loading control. (b) Quantification of ACE2 expression normalized to β‐actin. Data are presented as mean ± SD, with individual biological replicates shown (n = 3). ANOVA with post hoc tests: **p < 0.01. (B) ACE2 mRNA levels are detected by real‐time qRT‐PCR and calculated by the 2−ΔΔCT method relative to GAPDH. Data are presented as mean ± SD, with individual biological replicates shown (n = 7). ANOVA with post hoc tests: **p < 0.01. (C) Immunofluorescence staining of Calu‐3 cells. (a) ACE2 signal (red) in untreated cells. (b) Diminished ACE2 signal (red) after treatment with 200 µM UDCA for 48 h. (c) COVID‐19 spike (XBB.1.5) pseudovirus infection (green) in the absence of UDCA. (d) Reduced pseudovirus signal (green) in cells co‐incubated with 200 µM UDCA. Scale bars = 50 µm. (D) Merged immunofluorescence images. (a) Uninfected Calu‐3 cells showing ACE2 (red) without pseudovirus signal. (b) Pseudovirus‐infected…

FIGURE 3. UDCA modulates serum cytokine profiles in COVID‐19 patients. Serum cytokine concentrations are detected using the Bio‐Plex 200 System (Bio‐Rad) in UDCA‐free and UDCA groups on Day 0 (before antiviral and/or UDCA treatment), and Days 3 and 6 (after UDCA treatment). Individual data points, means, and SD are shown. (A and B) Representative cytokines showing no significant response to UDCA: (A) IL‐12p70 and (B) RANTES. (C–H) Representative cytokines showing significant response to UDCA: (C) CTACK and (D) IL‐2Rα are significantly reduced after 3 days of UDCA treatment; (E) IL‐18 is significantly reduced after 6 days of UDCA treatment; (F) eotaxin is significantly increased after 3 days of UDCA treatment; (G) IL‐6 and (H) TRAIL are significantly reduced after 3 days of UDCA treatment. UDCA‐free group: Day 0 (n = 53); UDCA group: Day 0 (n = 89), Day 3 (n = 89), and Day 6 (n = 89). ANOVA with post hoc tests and/or Student's t‐test: *p < 0.05, **p < 0.01. Complete data for all 46 cytokines are provided in Table 3.