Astragaloside IV attenuates inflammatory response mediated by NLRP‐3/calpain‐1 is involved in the development of pulmonary hypertension

Abstract Inflammation eventually leads to pulmonary arterial hypertension (PAH). Astragaloside IV(AS‐IV) has a protective effect on pulmonary hypertension, but the specific protective mechanism has been unclear until now. Therefore, in this study, our aim was to investigate the mechanisms underlying the effects of AS‐IV on PAH. In vivo, male Sprague‐Dawley (SD) rats were injected intraperitoneally with monocrotaline (MCT, 60 mg/kg) and treated with AS‐IV (40 mg/kg, 80 mg/kg), MCC950 and MDL‐28170. In vitro, human pulmonary artery endothelial cells (HPAECs) were treated with monocrotaline pyrrole (MCTP, 60 μg/mL). The protein expression levels of NLRP‐3, caspase‐1, ASC, IL‐18, IL‐1β and calpain‐1 were measured in vivo and/or in vitro. The results showed that AS‐IV decreased the protein expression levels of NLRP‐3, caspase‐1, ASC, IL‐18, IL‐1β and calpain‐1 in vivo and/or vitro. In conclusion, in this study the results suggested that AS‐IV could inhibit monocrotaline‐induced pulmonary arterial hypertension via the NLRP‐3/calpain‐1 pathway.


| Animal models and drug treatments
Male Sprague-Dawley rats weighing 180-200 g (Experimental Animal Center, Jinzhou Medical University, Jinzhou, China) were housed in cages with free access to food and water, and exposed to a 12-hour light/dark cycle at a controlled temperature (25 ± 2°C).
All animal treatment protocols used in this study were approved by the Animal Experimentation Ethics Committee of Jinzhou Medical University. After 1 week of pre-adaptation, the rats were randomly the rats were anesthetized with 20% urethane and then killed.

| Immunohistochemical staining
After dewaxing and antigen repair, the slides were eliminated peroxidase activity and blocked non-specific binding. The slides were incubated with primary antibodies for anti-NLRP-3 (1:100) at 4°C overnight, followed by incubation with an HRP-conjugated secondary antibody. Then, the sections were stained with DAB and counterstained with haematoxylin. Image analysis was measured by Image-Pro Plus software.

| Immunofluorescence
The slides were permeabilized with 0.1% Triton X-100 in PBS for 30 minutes and then incubated with 5% bovine serum albumin in PBS for 60 minutes. The slides were incubated with primary antibody against anti-calpain-1 (1:100) at 4°C overnight. Subsequently, the slides were incubated with the fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody and Hoechst 33258 staining solution.
Image analysis was performed using Image-Pro Plus software.

| Enzyme-linked immunosorbent assay (ELISA)
The contents of IL-18 and IL-1β in serum were detected according to the manufacturer's instructions for each kit.

| Western blotting
The collected lung tissue and HPAECs were homogenized in RIPA lysis buffer. The protein concentrations were measured using a BCA protein assay kit. The samples were separated by SDS-PAGE (10%-12% polyacrylamide gel) and transferred to a PVDF membrane, which was blocked with 1% BSA for 1.5 hours and then incubated with antibodies calpain-1, NLRP-3, caspase-1, ASC, IL-18, IL-1β, GAPDH and β-actin overnight at 4°C. The membrane was washed three times with TBST and then incubated at room temperature with an HRP-conjugated secondary antibody for 2 hours. The results were analysed with ImageJ software.

| Data analysis
The data are presented as the mean ± SD for normally distributed data and were analysed with SPSS 25.0. The data were processed with one-way ANOVA. One-way ANOVA with post hoc tests was performed for multiple-group analysis. P < .05 was considered statistically significant.

| D ISCUSS I ON
The assembly of the NLRP-3 inflammasome can lead to the activation of caspase-1. 12 Caspase-1 is activated by proximity-induced autocatalytic recruitment to the inflammasome. Active caspase-1 cleaves the inflammatory factors pro-IL-18 and pro-IL-1β to generate mature IL-18 and IL-1β. Therefore, we tested whether AS-IV could affect the NLRP-3 activation pathway. Our data show that AS-IV can inhibit the expression of NLRP-3, caspase-1, ASC, IL-18 and IL-1β in vivo and in vitro. Our study shows that NLRP-3 is involved in regulating the expression of IL-8 and IL-1β in rats with pulmonary hypertension. In a recent report, Western blotting analysis confirmed that caspase-1 activation and IL-1β maturation in calpain-1-deficient cells were blocked. 13 In addition, calpain inhibition or silencing attenuated myocardial ischaemia-reperfusion injury in mice through the NLRP-3/ASC/caspase-1 axis. 14 Therefore, we suggest that the inflammatory response mediated by NLRP-3/calpain-1 is involved in the development of pulmonary hypertension.
We used the NLRP-3 inflammasome inhibitor (MCC950) and the calpain-1 inhibitor (MDL-28170) to verify that AS-IV protected rats against pulmonary hypertension induced by MCT through the NLRP-3/calpain-1 signalling pathway. For the first time, we observed the improvement of the inflammatory response mediated by NLRP-3/calpain-1 as a breakthrough that could scientifically explain the prevention and treatment effects of AS-IV on pulmonary hypertension. In addition, the pathogenesis of the underlying disease was mainly discussed from the perspective of the improvement of the effects on endothelial cells. However, the mechanism underlying the protective effect of AS-IV on MCT-induced pulmonary hypertension in rats mediated by the NLRP-3/calpain-1 signalling pathway is not clear. Therefore, we will generate calpain-1 knockout mice to further clarify the specific mechanism.

ACK N OWLED G EM ENTS
The author would like to thank Professor Hongxin Wang, and the senior brothers and sisters of the laboratory. This work was supported by National Natural Science Foundation of China (81973553). F I G U R E 2 AS-IV inhibited the activation of the NLRP-3/calpain-1 pathway in MCP-induced HPAECs. The protein expression levels of NLRP-3, caspase-1, ASC, IL-18, IL-1β and calpain-1 in HPAECs were measured by Western blotting (A-G). The data are presented as the mean ± SD (n = 3; ## P < .01 vs Control group, **P < .01 vs MCTP group)