Andrographolide Suppresses Expressions of Coagulation and Fibrinolytic Inhibition-Related Factors in LPS-Induced Alveolar Epithelial Cell Type II via NF-κB Signal Pathway In Vitro

Background Andrographolide (Andro) has been confirmed to ameliorate alveolar hypercoagulation and fibrinolysis inhibition via NF-κB pathway in acute respiratory distress syndrome (ARDS), but the specific target of Andro is unknown. Purpose Our aim is to explore the specific target of Andro through which the drug exerted its effects on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS. Methods AECII was treated with different doses of Andro for 1 h, and then stimulated with LPS for 24 h. Expressions of tissue factor (TF), plasminogen activator inhibitor (PAI)-1 and tissue factor pathway inhibitor (TFPI) were detected. Concentrations of thrombin-antithrombin complex (TAT), pro-collagen type III peptide (PIIIP), antithrombin III (ATIII) and activated protein C (APC) in cell supernatant were measured by enzyme linked immunosorbent assay (ELISA). NF-κB signaling pathways activation was simultaneously determined. AECII with p65 down-/over-expression were used as control. Results Andro effectively inhibited TF and PAI-1 and promoted TFPI expressions on AECII induced by LPS stimulation. Andro also significantly suppressed the productions of TAT and PIIIP but promoted ATIII and APC secretions from the LPS-treated cell. Furthermore, Andro application obviously inhibited NF-κB signaling pathway activation provoked by LPS, as shown by decreased level of phosphorylation (p‑)-IKKβ/IKKβ, p-p65/p65 and p65 DNA binding activity. The effects of Andro on those factors were obviously strengthened by down- but were weakened by up-regulation of p65 gene in AECII cell. Conclusions Our data demonstrates that targeting AECII is the mechanism by which Andro ameliorates alveolar hypercoagulaiton and fibrinolytic inhibition via NF-κB pathway in ARDS. Andro is worth to be clinically further studied in ARDS treatment.


Introduction
Acute respiratory distress syndrome (ARDS), a common cause of death in intensive care units [1,2], is a devastating clinical syndrome characterized by non-cardiogenic pulmonary edema, respiratory distress and hypoxemia [3][4][5]. Although some progresses were made in the treatment in ARDS, its mortality is still as high as 35-45% [6]. The complex pathogenesis of ARDS is mainly responsible for the refractoriness and high mortality in this disease. Our previous studies have shown that LPS-induced ARDS exhibited alveolar hypercoagulability and fibrinolytic inhibition [7], which are important characteristics of this disease [8]. Alveolar hypercoagulation and fibrinolytic inhibition causes a large number of microthrombosis in the pulmonary blood vessels, massive fibrin deposition in alveolar cavity, lung tissue fibrosis, etc., which are associated with reduced lung compliance, V/Q mismatch and with refractory hypoxia in ARDS [9]. Therefore, it is pivotal to explore effective drugs for alveolar hypercoagulation and fibrinolytic inhibition in this disease.
Our previous studies confirmed that NF-κB pathway participates the regulation of alveolar coagulation and fibrinolysis inhibition in ARDS, and alveolar epithelial cell type 域 (AEC域) was testified to be a main effector cell responsible for these pathophysiologies [10][11][12]. Therefore, targeting AEC域 cell could be efficacious for ARDS treatment.
Andrographolide (Andro) is the main active components of the natural plant andrographis paniculata. It owns many pharmacological effects, including anti-inflammation, antibacterial, anti-virus, liver protection and anti-vascular properties [13][14][15][16][17]. In our previous animal study, Andro was confirmed to effectively ameliorate alveolar hypercoagulation and fibrinolytic inhibition via NF-κB signal pathway [18], but the specific target cell of Andro is unknown. Given that AEC域cell has important regulatory role in alveolar hypercoagulation and fibrinolytic inhibition in ARDS, so we observed the efficacies of Andro on expressions of coagulation and fibrinolytic inhibition-related factors in AECII cell under LPS stimulation.

Detection of Andro Cytotoxicity by Using Cell Counting Kit-8 (CCK-8)
In order to determine the optimal concentration of Andro for RLE-6TN cells, we used CCK8 to detect Andro cytotoxicity. RLE-6TN cells were seeded into a 96-well plate (5 × 10 3 cells/well in 100 μl volume) and pre-incubated for 24 h at 37 °C with 5% CO 2 . Different concentrations of Andro (0, 3.125, 6.25, 12.5, 25, 50, 100 µg/ml) were added into the wells. PBS was used as a blank control. The cells were cultured for 24 h in the incubator, and CCK-8 reagent (Dojindo Molecular Technologies, Inc.) was added at 10 μl/ well for 1 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader.

Experimental Protocol
According to the results of Andor cytotoxicity (Fig. 1), three different dosages of Andro, i.e., 6.25 µg/ml, 12.5 µg/ml and 25 µg/ml, were selected for the subsequent experiment. The cells were divided into five groups, LPS, Andro (subdivided into 6.25 µg/ml, 12.5 µg/ml and 25 µg/ml groups) and control (CN). In LPS group, cells were stimulated with 50 µg/ml LPS for 24 h, while the cells in Andro groups were co-cultured with different concentrations of Andro (Sigma, USA) for 1 h first and then were stimulated with a same dosage of LPS for another 24 h. There were no any manipulations for cells in control group (CN).

Down-(p65 −/− ) and Over-(p65 +/+ ) Expressions of p65 Gene Construction
In order to explore whether the mechanism of Andro is associated with NF-κB pathway, we constructed different levels of p65 expressions in the cell. Based on the p65 gene (Gene ID: 309165), we made cell lines with down-and overexpressions of p65 gene respectively by using the lentivirus transfection according to our previous method [11]. And then these cells were also treated by LPS with or without Andro administration.

Reverse Transcription-Quantitative (RT-q) PCR
The mRNA expressions of TF, PAI-1 and TFPI were detected by RT-qPCR. GAPDH was used as an internal reference. Briefly, cells were collected, and total RNA was extracted using TRIzol ® reagent (Takara Bio, Japan). RNA concentration was assessed using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). The A260/ A280 ratio of the extracted RNA was adjusted to 1.8-2.0. The primer sequences used were as follows: The reactions were set up as follows: 5 μl SYBR Green mix (Takara Bio, Japan), 0.5 μl forward primer, 0.5 μl reverse primer, 1 μl cDNA template and 2.8 μl ddH 2 O, for a total reaction volume of 10 μl. The entire reaction system was pre-heated at 95 °C for 30 s. qPCR was then performed using the folling thermo-cycling procedure: 95 °C for 5 s, 60 °C for 34 s and 95 °C for 15 min, 60 °C for 1 min, 95 °C for 15 s for 40 cycles. After that, dissolution and amplification curves of the target gene were recorded following gene amplification. Specificity of the reaction was evaluated, and the Ct value was calculated according to the dissolution and amplification curve. Expressions of target genes were calculated using the 2 −ΔΔCt method, where ΔΔCt = (Ct, sample target-Ct, sample GAPDH)-(Ct, control target-Ct, control GAPDH).

ELISA Assay
Cell supernatants and nuclear extract were harvested and stored at − 80 °C. Thrombin antithrombin (TAT), procollagen III propeptide (PIIIP), antithrombin III (ATIII), activated protein C (APC) (Shanghai Fanke Industrial Co., Ltd, China) and p65 DNA binding (Cayman Chemical, USA) were determined by using test kits according to the manufacturers protocol.

Statistical Analysis
Data are presented as the mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Tukey's post hoc test. p < 0.05 was considered to be statistically significant.

Andro Inhibits TF and PAI-1, but Promotes TFPI mRNA and Protein Expressions in LPS-Stimulated RLE-6TN Cells
LPS stimulation promoted TF, PAI-1, but inhibited TFPI expressions either in mRNA or in protein level in RLE-6TN cells, which were all reversed by Andro in dosedependent manner. (Fig. 2).

Andro Inhibits TAT and PIIIP but Promotes ATIII and APC Secretions from LPS Stimulated RLE-6TN Cells
The secretions of thrombin antithrombin complex (TAT) and procollagen peptide type III (PIIIP) significantly were seen to increase but that of activated protein C (APC) and of antithrombin III (ATIII) to decrease from LPS stimulated RLE-6TN cells. Andro application, however, significantly reversed the changes of all factors above in dose-dependent manner. (Fig. 3).

p65 Gene Significantly Influences Andro's Effect on Coagulation and Fibrinolysis Inhibition-Related Factors in RLE-6TN Cells
In order to further confirm whether the mechanism of Andro is associated with NF-κB pathway, we constructed cell lines with different p65 gene expressions. Our data interestingly showed that the efficacies of Andro on coagulation and fibrinolytic inhibition-related factors was tremendously enhanced with down-regulation of p65 (p65 −/− ) ( Fig. 6 and Fig. 7) but was obviously weakened with up-regulation of p65 gene (p65 +/+ ) ( Fig. 8 and Fig. 9).

Discussion
Alveolar epithelial cell type II (AECII) plays a pivotal role in the regulation of alveolar hypercoagulation and fibrinolytic inhibition in ARDS [10][11][12]. So we chose rat AECII cell line RLE-6TN as the experimental object. Gram negative bacterial infection such as bacteria pneumonia is the common cause of ARDS, and LPS is the most pathogenic factor of bacteria. So LPS is often used to replicate ARDS models in vitro and in vivo [19,20]. According to our previous studies, 5 mg/l of LPS was selected to stimulate the cell [10,11]. In ARDS process, alveolar hypercoagulation and fibrinolytic inhibition have been confirmed to be pivotal pathophysiologies, which mainly contribute to the refractory hypoxemia in ARDS. And our published data showed that alveolar epithelial cell type II (AEC II) essentially regulates coagulation and fibrinolytic inhibition by expressing coagulation and fibrinolytic inhibition related factors [10,11], which was confirmed again in this study in vitro.
As described in our previous researches [7,10], TF, PAI-1, TAT and PIIIP are associated with coagulation and fibrinolytic inhibition, while TFPI, APC and ATIII are anti-coagulators. So we selected these factors again as our observational targets. Our data showed that AEC 域-mediated hypercoagulation and fibrinolytic inhibition happened in condition of LPS stimulation. However, Andro effectively reversed the changes of above coagulation and fibrinolytic-related factors in AECII cell induced by LPS stimulation, which demonstrated that Andro has obvious efficacies on procoagulation and fibrinolytic inhibition mediated by AECII cell under LPS provocation. Fig. 4 Effects of Andro on NF-κB signal pathway activation induced by LPS. Western blotting was performed to measure total p65, p-p65, total IKKβ and p-IKKβ protein expression in cells, GAPDH was used as an internal control for protein reference. Compared with group NC, a p < 0.05; Compared with group LPS, b p < 0.05; Compared with group Andro 6.25, c p < 0.05; Compared with group Andro 12.5, d p < 0.05. A, B Western-blot bands of p65, p-p65, total IKKβ and p-IKKβ respectively. C Relative protein expression of p-p65. D Relative protein expression of p-IKKβ. p-p65 phosphorylated p65, p-IKKβ phosphorated IKKβ, Andro andrographolide   TAT thronbin-antithronbin complex, IIIP procollagen peptide type III, ATIII antithrombin III, APC activated protein C, Andro andrographolide NF-κB pathway was confirmed to regulate alveolar hypercoagulation and fibrinolytic inhibition in ARDS in our research [7,10]. Therefore, we explored whether this pathway is associated with the mechanism through which Andro exerts its efficacy on AECII cell mediated coagulation and fibrinolytic inhibition. Our results indicated that Andro significantly inhibited the LPS-stimulated NF-κB pathway activation, as shown by decreases of p-p65/p65 and p-IKKβ/IKKβ, and of p65 DNA binding activity. To further confirm the Andro's mechanism, we control the p65 expression. Data indicated that Andro's effects was obviously enhanced by down-regulation of p65 (p65 −/− ) but was remarkably weakened by up-regulation of p65 (p65 +/+ ). So, we have the reason to think that Andro Interestingly, as happened in our animal study [18], the effects of Andro became more obvious when dosage of the drug from 6.25 to 25 µg/ml, suggesting a dose-dependent manner.
From the results of current cell experiment and previous animal study, it is worth to further explore the value of Andro in the future.

Conclusion
Andrographolide ameliorates LPS induced expressions and secretions of procoagulant and fibrinolytic inhibitory factors in AECII through inactivation of NF signaling pathway. Our findings suggest the potential protective role of Andrographolide in alveolar hypercoagulation and fibrinolytic inhibition in ARDS.
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