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.