Isoliquiritigenin attenuates LPS-induced acute kidney injury through suppression of HMGB1 pathway in renal tubular against ferritinophagy CURRENT POSTED

Septic acute kidney injury (AKI) mainly results in life-threatening renal dysfunction involving renal tubular injury to bring heavy burden to patients in intensive care unit (ICU). However, there is still a lack of therapy to prevent septic AKI effectively and inexpensive. To observe the role and novel mechanism of isoliquiritigenin (ISL) which isolated from the roots of licorice in septic AKI, we used LPS to induce renal tubular injury upon septic AKI both in vitro and in vivo. 50mg/kg ISL and 5 mg/kg Ferrostatin-1 were once given to the male C57BL/6 mice one hour before 1 mg/kg LPS i.p injection. 50 μM and 100 μM ISL respectively pre-treat the human renal tubular cells 5 hrs before 2 μg/ml LPS stimulation. We found ISL pretreatment apparently reversed LPS-induced renal dysfunction and ameliorated murine renal tubular injury by suppression HMGB1 pathway. Furthermore, we observed that LPS induced autophagy and ferroptosis in renal tubular, whereas ISL pretreatment significantly suppress autophagy and ferroptosis of renal tubular both in vitro and in vivo. Mechanically, autophagy activated ferroptosis via NCOA4-mediated ferritinophagy. Moreover, HMGB1 is required for ferritinophagy in renal tubular. ISL treatment inhibited the expression of HMGB1. Taken together, these results suggest that ISL protects LPS-induced acute kidney injury through suppression of HMGB1 pathway in renal tubular against ferritinophagy.

Ferroptosis, a form of iron-dependent cell death, is identified by accumulated lipid peroxidation and iron 5 . Autophagy is a conversed degradation pathway that maintains cellular homeostasis 6 . ferritinophagy defied as an autophagic phenomenon and degraded via autophagy to release iron 7,8 .
In current study, nuclear receptor coactivator 4 (NCOA4) is the cargo receptor for ferritinophagy enriched in autophagosomes 7 . So, we are interested whether ferroptosis and ferritinophagy have a relationship.
High mobility group box 1(HMGB1), as a proinflammatory mediator, releases by damaged cells in inflammatory diseases 9 . Importantly, HMGB1 plays a significant role in sepsis-associated acute kidney injury 10 . As for mechanism, previous works demonstrated that HMGB1 involves in autophagy through various pathways 11 − 13 . But there is a gap between HMGB1 and iron-related cell death. Hence, we considered the potential mechanism that HMGB1 involved in autophagy, ferritinophagy and ferroptosis in LPS-induced AKI.
Isoliquiritigenin (ISL) is one of the bioactive components isolated from the roots of a Chinese traditional herb called licorice 14 . ISL exerted useful biological activities such as anti-inflammatory, anti-oxidative, anti-nephritic and anti-cancer activities 15 . Meanwhile, ISL treatment decreased nitric oxide (NO) production and inhibit autophagy process 14,16 . Moreover, ISL inhibited amount of reactive oxygen species (ROS) production 17 .
In this study, we have now explored the role of autophagy, ferritinophagy and ferroptosis in AKI induced by LPS. Mechanically, autophagy activated ferroptosis via NCOA4-mediated ferritinophagy in septic AKI.

Animal Model
All procedures were conducted in accordance with National Institutes of Health guidelines for animal care and use, Male C57BL/6 mice (six to eight-week-old, 22-25 g) from the Experimental Animal Center of Sichuan Provincial People's Hospital were treated with a standard laboratory diet. LPS was dissolved in normal saline and ISL was dissolved into 0.5% Tween-20/saline. AKI mice were given i.p.
with LPS. All the mice were randomly divided into six groups (n = 8): control, ISL, Fer, LPS, LPS plus ISL and LPS plus Fer group. LPS was i.p. injected at a dosage of 10 mg/kg. For ISL treatment, 50 mg/kg ISL was given to the mice via gavage before LPS injection. Fer (Ferrostatin-1, SML0583, Sigma-Aldrich, USA) dosed intraperitoneally with 5 mg/kg to mice. Mice were sacrificed by cervical dislocation at 8 hrs after LPS injection. At the same time, the kidney and serum samples were collected. The volume of blood samples was limited, thus the samples were randomly chosen from each group for CREA, BUN, SOD, MDA and NO tests.

Immunohistochemistry
Formalin-fixed renal tissues were paraffin-embedded and cut into 2 µm thick. The sections were deparaffinized and rehydrated as routine protocols. Tissues were subjected to immunohistochemical staining for GPX4 (1:200, abcam, ab955) and xCT (1 µg/ml, ab64693, abcam). The process was conducted in strict accordance with the kit protocol. The images were taken by an Olympus Coolpixmicro digital camera from Japan. The level of NO, MDA and SOD were detected by NO assay kit (A012, Jiancheng, Nanjing, China), MDA assay kit (A003-1-2, Jiancheng, Nanjing, China) and SOD assay kit (A001-3, Jiancheng, Nanjing, China) following the manufacturer's instruction.

Statistics Analysis
The results were expressed as means ± standard deviation (SD). Statistical analysis was performed using Graph Pad Prism 5 software by one-way ANOVA with post hoc test. Newman-Keuls multiple comparison test was used to compare differences. Differences between groups were indicated statistically significant at P < 0.05.

ISL prevents renal tubular epithelial injury in LPS-induced AKI
To validate ISL can ameliorated renal dysfunction in LPS-induced AKI, animal model was generated in mice by the injection of LPS and confirmed by HE staining (Fig. 1A). After LPS injection, renal tubules had moderate/severe degree injury, compared with control group. ISL treatment revealed significant protective effect which could ameliorate the renal tubules injury into mild degree in kidney tissue. production decreased sharply. Additionally, more SOD production to maintain homeostasis after ISL treatment. As for nitrite, Fig. 2 showed two concentration of ISL could decrease NO. But there is a dramatical decrease after 100 µM ISL instead of 50 µM, in contrast to LPS.

ISL inhibited LPS-induced autophagy, ferritinophagy and ferroptosis both in vivo and vitro
Autophagy has been implicated in the pathogenesis of various diseases and biological processes.
However, whether autophagy is injurious or protective for AKI has not clearly clarified. To validate the function in our model, we detected the most important markers which contribute to autophagy, including ATG7 and Beclin-1. The results showed ATG-7 and Beclin-1 were all elevated after LPS treatment, whereas ISL could inhibit these makers of autophagy both in vivo and vitro (Fig. 3).
Ferritinophagy is newly discovered selective autophagy. Overactivated ferritinophagy could induce cellular overload iron and then might participate in ferroptosis, so we examined the expression of NCOA4 which recognized as a specific carrier for ferritinophagy. We found LPS could trigger NCOA4 expression. After ISL treatment, NCOA4 expressed less in contrast to LPS group both in vivo and vitro.
More precisely, 100 µM ISL in LPS-induced cells showed a significant decreased accumulation of NCOA4 (Fig. 4). GPX4 is identified as the most highly ranked candidate of biomarker in ferroptosis 18 .
To examine the involvement of ferroptosis, we used western blot to show that GPX4 decreased in LPSinduced AKI which followed as autophagy biomarker. ISL can contribute to the accumulation of GPX4 both in normal and septic model. We highly considered that ferroptosis in LPS-induced AKI is an autophagy-related process.
HMGB1 might be a potential regulator of ISL in autophagy-related ferroptosis.
HMGB1 is required for LPS-induced autophagy in renal tubular. In the model of septic AKI, we found GPX4 decreased markedly, indicating that ferroptosis participated in this disease process.
Interestingly, HMGB1 expression had the same trend as GPX4 (Fig. 4). The results suggested that HMGB1 involved in autophagy-related ferroptosis. But HMGB1 expression was blocked after ISL treated, which revealed ISL promoted abundant GPX4 to maintain the cellular homeostasis by suppressing HMGB1.

Discussion
AKI is a multifactorial renal disease characterized by a rapid decrease of renal function, resulting in the accumulation of toxins and failure of other organs 19 . Clinically, sepsis and ischemia-reperfusion injury (IRI) are the main causes of AKI 19 . The effective intervention in the early stage needs to be found. ISL, isolated form the root of licorice, has studied protective effect in multiple disease.
Researchers found biological activities such as anti-inflammatory, anti-oxidative, anti-nephritic and anti-cancer activities 15 .
In our study, we established LPS-induced AKI model both in vivo and in vitro. We used the renal tissue from mouse model to do the H&E staining and found LPS injection leaded the renal tubules to serious damage. However, ISL treatment can attenuate the injury in pathology. In the previous study, we confirmed that ISL can decline the CREA and BUN to ameliorate renal function of LPS-induced AKI 20 .
These results showed the preliminary protective effect of ISL.
But the mechanism needs to be clarified. Multiple mechanisms participate in LPS-induced AKI. Above these results, we considered that ferroptosis might involve in LPS-induced AKI and the potential mechanism of ISL.
Autophagy has been implicated in the pathogenesis of various diseases and biological processes.
However, whether autophagy is injurious or protective for AKI has not clearly clarified. To validate the function in our model, we detected the most important markers which contribute to autophagy, including ATG7 and Beclin-1. We demonstrated autophagy exist in LPS-induced AKI, whereas ISL could inhibit autophagy both in vivo and vitro. R. Kang et.al considered Beclin-1 is a key regulator of autophagy, can promote ferroptosis through a Beclin1-SLC7A11 complex 22 . The study showed that ferroptosis can be inhibited when knockout ATG7 23,24 . Torii S et al. validated that autophagy is required for the induction of ferroptosis both in normal and cancer cells 25 . Above these studies, we inferred the ferroptosis in LPS-induced AKI is autophagy-mediated.
Ferritinophagy is newly discovered selective autophagy. Overactivated ferritinophagy could induce cellular overload iron and then might participate in ferroptosis. NCOA4 recognized as a specific carrier for ferritinophagy. We found LPS could trigger NCOA4 expression. After ISL treatment, NCOA4 expressed less than control group. Glutathione peroxidase (GPX4), an antioxidant enzyme and an inhibitor of selective ferroptosis, is highly expressed in NCOA4-null mice 26 . NCOA4-mediated ferritin degradation is involved in ferroptosis 6 . Torii S et al. also demonstrated autophagy can contribute to ferroptosis by degradation of ferritin 25 . Thus, we highly considered that autophagy activated ferroptosis via NCOA4-mediated ferritinophagy (Fig. 5). Further, HMGB1 is a DAMP released by ferroptotic cells in an autophagy-dependent manner 23 . In our study, HMGB1 expression had the same expression trend as GPX4, indicating HMGB1 expression might from ferroptosis.

Conclusions
In this study, we verified the anti-inflammatory effects of ISL in LPS-induced acute kidney injury both in vitro and vivo. Moreover, we found the mechanism was associated with ferroptosis and ferritinophagy. Besides, we considered the suppression of HMGB1 in renal tubular against ferritinophagy was implicated in AKI. Ferrostatin-1, an inhibitor of ferroptosis, showed its protective function in vivo. However, further studies are awaited to confirm this preliminary outcome. Dr. Yi Li host or participate in these funding mentioned above. All the funding to some extent had contribution to reagents purchase, experimental devices and consumables, animal and cell line culture, et al.

Author Contributions
YT completed the main experiments, and was a major contributor in writing the manuscript. YW, CW, MY, SC assisted experiments, and the order of them were followed by their experimental workload. LL contributed to IHC. GL and LW made contribution to analyzing the result of experiment and provide instructions. MX and YL designed this work. YL also provided funding to complete this study. All authors read and approved the final manuscript.   and C were tested in vitro and D, E and F were in vivo. * means comparation with LPS group and P < 0.05. ** means comparation with LPS group and P < 0.01. *** means comparation with LPS group and P < 0.001. # means comparation with control group and P < 0.05. ## means comparation with control group and P < 0.01.### means comparation with control group and P < 0.001.  ISL attenuates LPS-induced ferritinophagy involving HMGB1. LPS activated NCOA4 but suppressed HMGB1 and GPX4. With ISL treatment, the expression of NCOA4 was reduced and HMGB1 and GPX4 were increased.

Figure 5
The mechanism diagram of ISL. We found ISL suppressed autophagy-mediated ferritinophagy resulting in inhibition of ferroptosis and the pathway involved HMGB1 which might be a potential regulator.

Supplementary Files
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