Forsythoside A protects against lipopolysaccharide-induced 1 acute lung injury through up-regulating microRNA-124 2

Acute lung injury (ALI) is a life-threatening disease without effective pharmacotherapies, so far. Forsythia suspensa is frequently used in the treatment of lung infection in traditional Chinese 25 medicine. In searching for natural anti-inflammatory components, the activity and the underlying 26 mechanism of Forsythoside A (FA) from Forsythia suspensa were explored. In this paper, 27 BALB/c mice and murine RAW 264.7 cells were stimulated by LPS to establish inflammation 28 models. Data showed that FA inhibited the production of TNF-α and IL-6 and the activation of 29 STAT3 in LPS-stimulated RAW 264.7 cells. Additionally, FA increased the expression level of 30 miroRNA-124 (miR-124). Furthermore, the inhibitory effect of FA on STAT3 was counteracted 31 by the treatment of miR-124 inhibitor. Critically, FA ameliorated LPS-induced ALI pathological 32 damage, the increase of lung water content and inflammatory cytokine, cells infiltration and 33 activation of the STAT3 signaling pathway in BALB/c mice. Meanwhile, FA up-regulated the 34 expression of miR-124 in lungs, while administration with miR-124 inhibitor attenuated the 35 protective effects of FA. Our results indicated that FA alleviates LPS-induced inflammation 36 through up-regulating miR-124 in vitro and in vivo . These findings indicate the potential of FA 37 and miR-124 in the treatment of ALI.


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For the total protein extraction, RAW 264.7 cells were homogenized in lysis buffer (50 mM 130 Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF, 1mM Na 3 VO 4 , 1 mM 131 dithiothreitol, 1 mM phosphatase inhibitor) on ice for 15 min and vortexed for 45 s. Samples were 132 centrifuged at 15 000 xg at 4 o C for 20 min and the supernatants were harvested and stored at -20 and β-actin (for IL-6 and STAT3) were used as internal controls. All qPCR reactions were 155 performed using LightCycler ® 96 real-time PCR system (Roche, Basel, Switzerland), and the ΔΔ Ct 156 method was used for the data analysis. 157 158

Dual Luciferase Reporter Assay 159
The 3'UTR sequence of STAT3 was subcloned into the pRT-TK vector. Renilla luciferase 160 vector (pRL-SV40) was used to normalize the luciferase activity. Cells were infected by an 161 adenovirus carrying the pRT-TK vector or Renilla luciferase vector. After cells were incubated 162 with FA for 36 h, luciferase activity was determined using the Dual  The freshly harvested lung tissues were excised, blotted dry, and weighed by an electronic 212 balance (Sartorius, Göttingen, Germany) to obtain the wet weight (W). Then, the samples were 213 dried in a thermostatic dryer box (FUMA, Shanghai, China) at 60 o C. Until the weight was not 214 changed, the dry weights (D) were recorded. To assess the degree of pulmonary edema, W/D ratio 215 was determined by the following formula: W/D × 100 %. 216

Protein levels and cell count in BALF 218
After sacrifice of mice, the trachea was surgically exposed in the ventral neck area. The plastic 219 cannula was inserted into the trachea. BALF was performed with two equals of 0.5 mL of cold 220 NaCl solution (0.9%) instilled up to a total volume of 1.0 mL. BALF was centrifuged at 500 xg 221 for 10 min at 4 o C. The protein concentration in the supernatant was measured by the BCA protein 222 assay kit. The cell pellet was resuspended in 50 μl of NaCl solution and total BALF cells were 223 counted double-blindly with a hemacytometer (Sigma-Aldrich). 224 225

Statistical analysis 226
Data were presented as mean ± SEM of at least five independent experiments. ANOVA was 227 used to assess differences between multiple groups by SPSS 20.0 (IBM, Armonk, USA). Tukey's 228 test was used for multiple comparisons. P ≤ 0.05 were considered as statistically significant. 13 Previous research has reported that miRNAs are able to induce degradation of target mRNA or 262 inhibiting its translation process to regulate inflammation. Therefore, we next identified which 263 miRNA regulates STAT3 after the treatment of FA. The publicly available bioinformatics tools 264 (TargetScan, miRTarBase and miRDB) showed that there are 9 miRNAs, including miR-124, 265 miR-125a-5p, miR-130b, miR-874, miR-519d, miR-20b, miR-181a-5p, miR-17 and miR-20a, 266 targeting to STAT3 ( Figure 3A). Among these miRNAs, miR-124 has been reported to be a 267 regulator of STAT3 and is associated with the release of IL-6 [23]. Therefore, we tested the 268 expression of miR-124 in RAW 264.7 cells. We found that miR-124 was increased significantly 269 after FA treatment, and peaked at 36 h ( Figure  inflammation. MiRNAs, such as miR-100, miR-133a and miR-140 play an anti-inflammatory role 290 in ALI [24]. Therefore, we detected that whether FA could prevent ALI through up-regulating 291 miR-124. As shown in Figure 5A, B, FA or DEX reduced edema, hyperemia and inflammatory 292 cells infiltration in LPS-induced ALI mice. FA or DEX also down-regulated the total protein 293 ( Figure 5C) and the total cells ( Figure 5D) in BALF and lung W/D ratio ( Figure 5E). For 294 exploring the role of miR-124 in the anti-ALI effects of FA, miR-124 Antagomir (a miR-124 295 selective inhibitor) was used. According to our previous study, 10 μmol/kg Antagomir could 296 significantly inhibited the expression of miR-124 in the lung tissues ( Figure S2). Thus, Antagomir 297 at the dose of 10 μmol/kg was selected for the subsequent experiments. As shown in Figure 5, the 298 anti-ALI effects of FA were counteracted in the presence of miR-124 Antagomir. 299 We further detected the expression levels of miR-124 and related proteins in lung tissues. As 300 shown in Figure 6A -C, FA up-regulated the expression of miR-124, and suppressed the 301 phosphorylation of STAT3 at Tyr705 and the protein expression of STAT3. Moreover, FA 302 inhibited the release of IL-6 in BALF from ALI mice, which was similar to DEX ( Figure 6D). 303 However, these effects of FA could be counteracted by inhibition of miR-124. Collectively, FA 304 inhibited inflammatory responses in ALI mice through up-regulation of miR-124. 305 Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200598/893817/cs-2020-0598.pdf by guest on 26 September 2020

Discussion 307
Although treatment option and understanding of mechanism of ALI has made great progress, 308 the mortality rate is still remains steady, which approaches 40% [25]. anti-inflammatory effects through up-regulation of miR-124, and subsequently suppressing the 642 STAT3 signal pathway to decrease the release of IL-6. 643