Triptolide’s anti-inflammatory effects on ARDS by down-regulating miR-9-5p and up-regulating LRG1 and CLDN5


 Background

Acute respiratory distress syndrome (ARDS) is a life-threatening respiratory disease and its treatment is not fully established. Triptolide, one of Tripterygium wilfordii’s main active components, has been proved to alleviate Lipopolysaccharide (LPS)-induced ARDS. Imbalance of MicroRNAs (miRNAs) is recognized as the pathogenic mechanism of various diseases, including ARDS. However, the specific miRNAs that play a key regulatory role in the anti-inflammatory effect of triptolide in ARDS remain elusive.
Methods

In this study, we administered triptolide in a mouse model of ARDS, and whole transcriptome sequencing was applied to identify meaningful miRNAs and validate them in vitro.
Results

The results showed that triptolide may reduce the inflammatory response in ARDS by regulating miR-9-5p. The data further proved that LRG1 and CLDN5 expression are regulated by miR-9-5p, and triptolide can down-regulate the expression of miR-9-5p by regulating negatively the expression of LRG1 and CLDN5.
Conclusion

Our study revealed that miR-9-5p was the specific miRNAs that plays key role in triptolide’s alleviation of ARDS inflammation by regulating target genes, and its inhibitory effect on LRG1 and CLDN5 expression was verified.

Triptolide, a diterpene lactone in Tripterygium wilfordii, has anti-in ammatory, immunosuppressive, antitumor effects. Triptolide-treated mice exhibited signi cantly reduced leukocyte, myeloperoxidase (MPO) activity, edema of the lung, as well as TNF-a and IL-6 production in the bronchoalveolar lavage uid, thereby attenuating an LPS-induced in ammatory response [5][6]. Triptolide is considered to be a promising potential therapeutic reagent for ARDS treatment.
Moreover, miRNAs are short single-stranded RNAs that regulate post-transcriptional mRNA expression by binding to complementary mRNA sequences, leading to translation suppression and gene silencing [7]. These miRNAs play critical roles in various physiological processes such as tissue development and differentiation, cell proliferation and tissue repair [8][9]. It is believed that miRNAs may be alternative therapies for many intractable diseases [10][11]. However, whether there are miRNAs that play a vital role in the secretion of proin ammatory cytokines has not been investigated.

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The aim of this study was to identify potential members of this subset that could serve as a new possible mechanism by which triptolide alleviates ARDS in ammation. Here, we report the identi cation of miR-9-5p, a miRNA previously unrelated to the phenomenon of ARDS, targeting two key mRNAs involved in ARDS: LRG1 and CLDN5.

Materials And Methods
Mice and Drugs C57BL/6 mice (female, 8 weeks old) were purchased from Institute of Animal Modeling, Nanjing University. Mice were intraperitoneally injected with 10mg/kg LPS(Sigma). The triptolide (Provided by Fujian Academy of Medical Sciences) was dissolved in 0.1% dimethyl sulfoxide (DMSO). The concentrations of triptolide L, M and H were 2.5ng/mL, 5ng/mL and 10ng/mL, respectively. The corresponding concentrations of L, M and H were 0.1mg/kg, 0.2mg/kg and 0.4mg/kg when intraperitoneally injected into mice.

Measurement of cytokines
Levels of IL-6, TNF-a and MCP-1 in the supernatant were analyzed using ELISA kits, and optical density (OD) values were determined using a multifunctional enzyme plate analyzer (Synergy 2, USA, Bio-Tek, Inc.).

Lung Wet to Dry Weight Ratio and Pathological score of lung
The lower lobe of the right lung was excised, and the wet weight (W) was measured after removal of the liquid on the surface of the collected lung tissue. Then, the lung tissue was placed in an oven at 80 °C for 24 h to obtain the dry weight, and the lung tissue W/D ratio was calculated by dividing the wet weight by the dry weight. lung injury scores were determined based on the following histological features, as described previously [12].

RT-qPCR and Complete transcriptome sequencing
The expression of miR-9-5p was determined using the TaqMan microRNA assay kit and SYBR green realtime polymerase chain reaction (ABI 7500 system). RT-qPCR primers are shown in Table.S1. The complete transcriptomes were sequenced with RNA sequencing (RNA-Seq) and miRNA sequencing (miRNA-Seq) (Beijing Aovesen Gene Technology Co., Ltd).
The target protein bands were visualized using Automatic chemiluminescence imaging system (Bio-Rad Lab) and quanti ed using Image-Pro Plus 6.0 software.

Statistical analysis
All data were analyzed using GraphPad Prism 8.0 software and represented as the mean SD of at least three experiments. The relationship between miR-9-5p and LRG1 and CLDN5 gene levels was determined by Spearman correlation analysis. p 0.05 was considered statistically signi cant.

Triptolide alleviates pulmonary in ammation in ARDS
The concentration of triptolide less than 10ng/mL had no signi cant cytotoxic effects on the BEAS-2B, and the amount of triptolide did not affect the survival of the mice (Fig.S1). As shown in Fig.1A, pretreatment with triptolide (L, M, and H) signi cantly decreased the lung W/D ratio compared with LPS group (p<0.01). Then, compared with the LPS group, the lung injury scores were reduced signi cantly in the triptolide group (Fig.1B) (P < 0.01). In addition, Fig.1C shows that triptolide decreased the expression of myeloperoxidase (MPO) mRNA in lung, while superoxide dismutase (SOD) mRNA expression increased After administration of triptolide. The levels of cytokines IL-6, TNF-a and MPC-1 were reduced in the bronchoalveolar lavage uid (Fig.1D-F, P<0.01). Based on these results, triptolide can help reduce the in ammatory response in ARDS.
Triptolide regulates miR-9-5p in ARDS Through complete transcriptome resequencing, the Gene Ontology (GO)and Pathway enrichment analysis of differential genes after triptolide intervention was mainly involved in substance exchange between membranes and the tight connection between cells, as is shown in Fig. 2A-B. Furthermore, 102 miRNAs were altered after treatment with triptolide (Fig. 2C), and these statistically signi cant changes could be visualized by using a re map (Fig.2D). Finally, differential miRNAs clustering analysis were used to determine the clustering patterns of differential miRNAs expression (7 miRNAs, including miR-9-5p) (Fig.2E).
LRG1 and CLDN5 expression are regulated by miR-9-5p As is shown in Fig.3A, the mRNA expression of miR-9-5p was signi cantly decreased after administration of triptolide in lung, compared with the LPS group. The MiRanda prediction software identi ed the putative binding sites (BSs) for miR-9-5p in the 3' untranslated regions (3'UTRs) of the LRG1 and CLDN5 mRNA, respectively (Fig. 3F).This prediction was functionally validated by over-expressing miR-9-5p in BEAS-2B cells, which resulted in a decrease of LRG1 and CLDN5 mRNA and protein levels ( Fig. 2B-E).
To determine whether miR-9-5p directly regulates LRG1 and CLDN5 expression by binding to their 3'UTR, putative WT and mutated 3'UTR were cloned into the luciferase reporter vector pmirGLO. Transfection with pre-miR-5p signi cantly decreased the luciferase activity of the reporter vector containing WT 3'UTRs while this reduction was reversed in the presence of miRNA inhibitor-9 ( Fig.3G-H). The decrease in the luciferase activity was completely reversed after point mutations (PM) in both miR-9-5p BSs in the 3'UTR of LRG1 and CLDN5 (Fig.3G-H). These observations indicated that BSs were critical for the regulation of LRG1 and CLDN5 3'UTRs, respectively. These data are consistent with a direct regulation of LRG1 and CLDN5 by miR-9-5p.

MiR-9-5p affected the changes of in ammation in vitro
To determine whether miR-9-5p was involved in the pro-in ammatory cytokines of ARDS by triptolide, BEAS-2B cells were transfected with pre-miR-9-5p and treated with triptolide. The transfection effect was veri ed by detecting the mRNA expression level of miRNA through RT-PCR (Fig. 4A). Increasing miR-9-5p levels signi cantly elevated the level of IL-6, TNF-a and MCP-1 (Fig.4B-D). Similarly, over-expression of miR-9-5p strongly reduced LRG1 and CLDN5 protein abundance (Fig.4E). Importantly, the reduction in the expression of LRG1 and CLDN5 observed in the presence of miR-9-5p was signi cantly restored after administration of triptolide (Fig.4E). These data suggested that triptolide can exert anti-in ammatory effects by targeting LRG1 and CLDN5 through miR-9-5p.

Discussion
In alleviating pulmonary in ammation of ARDS, miR-9-5p played a communication role in connecting triptolide, LRG1 and CLDN5. In other words, our study indicated that triptolide can down-regulate the expression of miR-9-5p, and miR-9-5p can regulate negatively the expression of LRG1 and CLDN5. More importantly, triptolide can reverse the negative regulatory effect of miR-9-5p, and then the expression level of LRG1 and CLDN5 tends to be normal.
ARDS is a common and serious clinical complication with high morbidity and mortality. The initiation and ampli cation of pro-in ammatory cytokines or mediators, such as IL-6, TNF-a and MCP-1 [13][14], are one of the factors that induce ARDS. Therefore, reducing the secretion of these in ammatory cytokines will signi cantly improve the clinical symptoms and prognosis of ARDS. Our study revealed that triptolide has a powerful anti-in ammatory effect, which signi cantly inhibits the expression of pro-in ammatory cytokines. MiRNAs have also been involved in the studies of triptolide, for example in the anti-tumor [15], anti-in ammatory [16] and immunosuppressive activities [17]. The quantitatively important change observed in miR-9-5p and the absence of information on this miRNA in the context of triptolide encouraged us to focus on miR-9-5p as a potential candidate susceptible of regulating LPS-mediated ARDS. Therefore, we screened out miR-9-5p and found that overexpression of miR-9-5p could increase the secretion of in ammatory cytokines such as IL-6, which further prompted us to explore target genes of miR-9-5p.
It is important that the involvement of LRG1 and CLDN5 in the pathogenesis of ARDS has also been reported [18][19]. LRG1 protein is a highly conserved member of the leucine-rich (LRR) protein family. LRG1 can also inhibit the secretion of in ammatory cytokines and pro-brotic cytokines [20], participate in epithelial cell proliferation, affect airway remodeling [21]. On the other hand, Membrane protein Claudin 5 (CLDN5) is an important component of endothelial tight junctions, which can maintain the normal barrier function of endothelial cells [22] and the integrity of endothelial tight junctions, and determine vascular permeability as well [23]. What's more, the destruction of pulmonary epithelial and endothelial cell barriers is the key to pulmonary edema caused by ARDS.
The results of our Complete transcriptome sequencing also focused on the substance exchange between membranes and the tight intercellular connection. Therefore, it can be inferred that triptolide may exert an anti-in ammatory effect by regulating LRG1 and CLDN5. Then, CLDN5 and LRG1 were predicted and veri ed in vitro. When we upregulated miR-9-5p, the expression of LRG1 and CLDN5 was decreased.
However, when we administered triptolide, the expression levels of LRG1 and CLDN5 were reversed, which veri ed that these two genes could play their protective role in ARDS and eventually reduce in ammation.
Although results revealed the regulatory effect between miR-9-5p and triptolide, Bioinformatics and other tools are still needed to synthesize the complex networks involved in triptolide's pharmacological activity. It is also necessary to further analyze the mechanism of triptolide's action on miR-9-5p in combination with the knowledge of drug structure analysis, and to study the speci c signaling pathway in vivo. At the same time, X-box binding protein1 (XBP1) should also be considered in the regulation of LRG1 and CLDN5 by triptolide, so as to further explore its speci c and detailed mechanism.

Declarations
WT 3′UTR sequence and pre-miR-9-5p co-transfected cells. Full-length blots are presented in Supplementary Figure 2. After the expression of miR-9-5p was changed in BEAS-2B, the contents of anti-in ammatory factors IL-6, TNF-a and MCP-1 were detected by ELISA. **P < 0.01 vs. Pre-miR-NC or miRNA Inhibitor-NC group. (E) After the expression of miR-9-5p was changed in BEAS-2B, Western blot analysis detected the expression levels of LRG1 and CLDN5 proteins. Data are shown as mean ± SEM; and **P < 0.01 vs. Pre-miR-NC or Triptolide + Pre-miR-9-5p group. Full-length blots are presented in Supplementary Figure 2.

Supplementary Files
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