4. DISCUSSION AND CONCLUSIONS
Although AKI and ALI are distinct entities, but they are frequently present simultaneously often inextricably linked in critically ill patients. Most intensive care unit patients will die from multiple rather than individual organ failure (Vincent, 2011). Sepsis is the leading cause of both AKI and ALI, and sepsis is characterized by systemic inflammation caused by infection (Yanet al. , 2019; Silveira et al. , 2021). LPS as a potent inducer of excessive inflammatory response, is widely used to establish AKI and ALI mouse models, which exhibit similar pathological changes to those found in human infectious sepsis (Ju et al. , 2018; Islam et al. , 2019). Currently, there are no effective strategies to prevent and cure AKI and ALI. Accumulating evidence suggests that anti-inflammation could be an effective therapeutic strategies for AKI and ALI treatment (Xie et al. , 2018; An et al. , 2019; Deng et al. , 2020).
NF-κB is involved in the gene expression of LPS-induced inflammation response and the pathophysiology in septic AKI and ALI. Attenuated NF-κB signaling has been shown previously to mediate protection against sepsis (Ibrahim et al. , 2020). Niu et al. showed that Harmine mitigates LPS-induced AKI through inhibition of the NF-κB signaling pathway (Niu et al. , 2019). Chi et al. found that Limonene attenuated the pulmonary inflammatory responses induced by LPS in ALI via suppression of the NF-κB and JNK pathways (Chi et al. , 2013). JNK is also an important mediator of inflammation that participated in the development of LPS-induced AKI and ALI. Additionally, Additionally, JNK can directly activate NF-κB by promoting IκB-α degradation, and JNK and NF-κB can coordinate the inflammatory response. Grynberg et al. confirmed that administration of JNK inhibitors can protect against AKI (Grynberg et al. , 2017). Zheng et al. verified that JNK inhibitor SP600125 alleviated LPS-induced ALI (Zheng et al. , 2014). Thus, NF-κB is an attractive therapeutic target for LPS-induced AKI and ALI. We designed and synthesized numerous NF-κB inhibitors, and screen their transcriptional inhibition activity using an NF-κB reporter assay. We found compound 270 could inhibit NF-kB transcription with IC50 values with 2.49 μM. CETSA assay indicated that 270 directly interacts with NF-κB proteins.
Macrophages are the principal immune cells mediator inflammation in kidney and lung tissues (Huang et al. , 2019). Thus, we examined the anti-inflammation activity of 270 in LPS-induced macrophages. We found that 270 inhibited the activation of NF-κB and JNK signaling pathways and expression of pro-inflammatory genes in LPS-induced RAW 264.7 macrophages and BMDMs, and 270 suppressed the secretion of TNF-α, IL-6 and MCP 1 in LPS-reduced BMDMs, suggesting that 270 possess the anti-inflammation activity in vitro.
We also verified that oral administration with 270 alleviated LPS-induced AKI and ALI in mice via improving inflammation in vivo. We discovered that 270 eliminated various pathological changes in renal and lung tissues, alleviated the enhanced of plasma Crea, BUN and pro-inflammation cytokines, and reversed the high expression of NGAL and KIM1 in kidney, accompanied by diminishing mRNA expression of pro-inflammation genes and blocking the activation of NF-κB and JNK signaling pathways in kidney and lung tissues. Beside, MPO is a biomarker of neutrophils and macrophages accumulation manifests severity of inflammation in tissues (Islam et al. , 2019; Jiang et al. , 2019), and 270 attenuated the elevated MPO activity in renal and lung tissues, indicating that 270 possess the anti-inflammation activity in vivo.
Increasing evidences imply that pathogenesis of LPS-induced septic AKI and ALI are multifactorial and complex, involving the interplay among inflammation, oxidative stress, ER stress and autophagy (Ryter et al. , 2015; Zeng et al. , 2017; Yan et al. , 2018). It has been well documented that NF-κB signaling pathway activation was also closely related to the progression of oxidative stress, ER stress and autophagy (Muriach et al. , 2014; Huang et al. , 2016; Islam et al. , 2019). LPS accelerated lipid-peroxidation-mediated cytotoxic MDA formation, which is one maker of oxidative stress and can further contribute to organs injury (Baradaran Rahimi et al. , 2019; Islam et al. , 2019). We found MDA content was enhanced in LPS-induced mice kidney and lung tissues consistent with reported results, but mitigated by 270.
LPS also can trigger ER stress, which becomes a major pathogenic factor in various diseases (Zeng et al. , 2017). Zhang et al. showed that MS-275 exhibits the nephroprotective effect on LPS-induced AKI by inhibiting ER stress (Zhang et al. , 2018a). Zeng et al. proved that 4-PBA ameliorates LPS-induced ALI through inactivating ER stress (Zeng et al. , 2017). In our study, we found BiP and CHOP, which were considered critical markers of ER stress, were elevated in LPS-induced mice kidney and lung tissues, and these alterations were restored by 270 treatment.
Studies have indicated that autophagy exerts a dual role in LPS-induced septic AKI and ALI (Zhang et al. , 2019). Mei et al. study showed that renal autophagy was rapidly induced by LPS, and inhibition of autophagy by chloroquine aggravated LPS-induced AKI in C57BL/6 mice (Meiet al. , 2016). But Zhao et al. pointed that dexmedetomidine protects against LPS-induced AKI by enhancing autophagy through inhibition of the PI3K/AKT/mTOR pathway (Zhao et al. , 2020). Zhang et al. found that the activation of autophagy in LPS-induced mice renal tissues, ACE2 activator RES could alleviate the severity of AKI via inhibiting autophagy and ACE2 inhibitor MLN-4760 aggravated the damage by promoting autophagy (Zhang et al. , 2019). However, Zhao et al. showed that LPS-induced ALI could be further exacerbated after suppression autophagy and enhancement of autophagy ameliorates ALI by weakening lung dysfunction (Zhao et al. , 2019). Both LC3A and p62 are usually used as biomarkers to monitor the level of autophagy, whose increased expression indicates the decreased autophagy (Song et al. , 2017). In our study, we discovered the inhibition of autophagy in LPS-induce mice kidney and lung tissues, as evidenced by the increment of LC3A and p62 protein levels, and pretreatment with 270 facilitated autophagy. The differences of these results in autophagy between we and others are likely associated with the use of various experimental models, animal background and the time-points monitored (Zhao et al. , 2020).
The development of LPS-induced AKI and ALI is not a mere singular mechanism, 270 alleviates LPS-induced septic AKI and ALI involving inflammation, oxidative stress, ER stress and autophagy, suggesting that 270 may be clinically viable. But several questions related to our findings remain to be answered. First, the detail mechanisms of 270 on oxidative stress, ER stress and autophagy need further study. Second, the cross-talk between inflammation, oxidative stress, ER stress and autophagy mediated by NF-κB signaling pathway require further elucidation. In addition, we found ALI induced by intraperitoneal injection of LPS was less severe than AKI. Despite ALI could be induced by intranasal, intratracheal, intraperitoneal or intravenous administration of LPS, whereas LPS caused acute pulmonary damage in mice 24 h after the intranasal or intratracheal administration, intravenous and intraperitoneal administration did not lead to a tissue-specific or similar degree of lung injury (Chenet al. , 2010). To further verify the effects of 270 on ALI, the protective effects of 270 in ideal animal models of ALI should also be investigated.
In conclusion, our results reveal that NF-κB inhibitor 270 protects against LPS-induced AKI and ALI primarily through inhibiting NF-κB and JNK-mediated inflammation response, as well as improving oxidative stress, suppressing ER stress and promoting autophagy (Figure 9), and supply evidence that 270 may be a valuable therapeutic medicine against inflammation-related diseases.