Our study explored the impact of cannabinoids on inflammation, apoptosis, and tissue damage in two ARDS models induced by ANTU (indirect lung injury) and LPS (direct lung injury). We found that JZL184 offers protection against tissue damage, inflammation, and apoptosis in the lung, modulating these processes. Additionally, variations in JZL184's protective effects were noted, aligning with the distinct pathophysiological mechanisms in the two ARDS models.
ARDS, marked by non-cardiac pulmonary edema, increased alveolo-capillary permeability, inflammation, fibrosis, resistant hypoxemia, decreased compliance, reduced functional capacity, and diffuse chest radiography infiltrates [1, 2], is studied using various animal models. In our previous studies, we demonstrated for the first time the roles of endothelin peptides [20], the L-arginine/nitric oxide (NO) pathway [21], lipid peroxidation [23], and inducible nitric oxide synthase (iNOS) expression [16] in ANTU-induced ARDS model and demonstrated that morphine [16], pentobarbital, thiopental, urethane [22], and dexmedetomidine [28] can mitigate its effects.
Recent findings suggest the endocannabinoid system's role in numerous diseases, positioning it as a potential therapeutic target [5]. The identification of CB1 and CB2 receptors, along with their primary ligands anandamide and 2-AG, and related enzymes, has deepened understanding of this system, which is of significant pharmacological interest due to its extensive distribution in mammals and regulatory role in various physiological functions, including immune response and inflammation [29, 30]. 2-AG and anandamide are broken down by MAGL and FAAH enzymes, respectively [31]. The endocannabinoid system, involving cannabinoid receptors and various metabolites, modulates immune cell functions and is prevalent in human lungs, with most cell types expressing cannabinoid receptors [32–34]. Research has shown the presence of CB1 and CB2 receptors in lung tissues, and many cells can produce endocannabinoids in reaction to inflammatory triggers, though their impact on lung health and disease remains to be fully elucidated. Studies have shown that the CB2 receptor agonist JWH133 can mitigate lung issues in conditions like RSV infection in human and mice [35], reduce interstitial lung fibrosis induced by nicotine [36] and ischemia-reperfusion-induced lung damage in mice [37], suggesting cannabinoids as promising agents for lung disease treatment.
In our study, the ANTU group exhibited more pronounced edema and perivascular enlargement compared to the LPS group, with non-hemorrhagic, exudative pleural effusion observed only in the ANTU group due to endothelial cell targeting, leading to significant pulmonary edema. Conversely, the LPS group showed limited edema as the primary damage was to the epithelium. Additionally, while the ANTU group had no inflammatory cells in the bronchiolar lumen, the LPS group displayed diffuse inflammatory cell presence, likely due to neutrophil migration following direct LPS administration, with LPS-induced vasodilation contributing to the noticeable congestion in the LPS group.
Lung diseases like ARDS, asthma, and bronchoalveolar dysplasia are linked to unregulated NF-κB activation, with ARDS-related endothelial cell dysfunction being notably influenced by NF-κB [38–40]. In our research, NF-κB staining was predominantly in the alveolar walls in the ANTU group and around the bronchiolar epithelium in the LPS group, indicating that endothelial damage drives ANTU-related injuries, while epithelial damage underlies LPS-induced injuries. The increased NF-κB staining in both injury models highlights the involvement of inflammatory mechanisms in lung pathology.
Apoptosis, a regulated cellular death process initiated through intrinsic or extrinsic pathways, leads to caspase-3 activation—known as the executioner caspase—following caspase-8 or caspase-9 activation [41, 42]. Caspase-3 orchestrates apoptosis by targeting various cell components. In our study, like NF-κB, caspase-3 staining was primarily seen in alveolar wall tissues in the ANTU group and near bronchiolar epithelium in the LPS group, indicating apoptosis's role in both endothelial and epithelial damage linked to indirect and direct lung injury mechanisms, respectively. The increased caspase-3 staining in pathology groups points to the significance of apoptotic mechanisms in lung pathology across both injury models.
JZL184 acts by inhibiting MAGL, which breaks down the endocannabinoid 2-AG, thereby elevating 2-AG levels [43] and exhibiting significant immunosuppressive and anti-inflammatory effects [44, 45]. Our results corroborate these findings. Similar to a study where a CB2 agonist (ABK5) reduced hindpaw edema in rats [46], we observed that JZL184 significantly reduced edema and perivascular dilatation in both injury models, and also lessened pleural effusion in ANTU-induced lung injury, suggesting cannabinoids modulate edema formation mechanisms.
JZL184 significantly reduced inflammatory cell presence in the ANTU group's interstitium and the LPS group's bronchiolar lumen, likely due to its effects on cell migration triggered by endothelial injury from ANTU and bronchiolar infiltration from LPS. It also improved desquamation in the LPS group's bronchiolar epithelium with condensed nucleated cells, indicating varied benefits of JZL184 in indirect and direct ARDS models.
JZL184 significantly elevated GSH levels, indicating its potential in reducing oxidative stress by enhancing antioxidant defenses in lung injury. It also diminished NF-κB and caspase-3 staining in both ANTU and LPS groups, with notable reductions in alveolar walls for ANTU + JZL184 and bronchiolar epithelium for LPS + JZL184. These findings suggest that JZL184 modulates inflammatory and apoptotic pathways in lung injury, contributing to its therapeutic effects. The differential modulation in indirect and direct ARDS models points to specific targeting of endothelium and epithelium, respectively.