Theacrine alleviates inflammation and lung injury in septic mice by mediating SIRT3

Purpose: To evaluate the effect of theacrine on sepsis progression and sepsis-induced lung injury and its mechanism of action. Methods: Lung injury model of lipopolysaccharide (LPS)-induced sepsis was made in the mice by injected with 3 mg/kg LPS dissolved in 50 μL PBS or only PBS (control group = 10). The remaining three groups (divided into ten mice per group) were administered theacrine (10, 20, and 40 mg/kg), by oral gavage 1 hour after LPS treatment. Hematoxylin and eosin (H&E) staining was performed to confirm the effect of theacrine on lung injury. Quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) were conducted to determine inflammatory and oxidative stress factors. TUNEL as well as immunoblot assays were carried to confirm its effect on apoptosis and mechanism of action. Results: Theacrine significantly improved lung injury as well as lung relief score in LPS-induced sepsis mice (p < 0.01) and it significantly reversed the increased levels of inflammatory cytokines in a dose-dependent manner in LPS-induced sepsis mice (p < 0.01), In addition, theacrine administration significantly reduced the increased intensity of ROS and MDA levels, and significantly improved the levels of SOD in lung tissues (p < 0.01). Furthermore, it improved LPS-induced apoptosis of lung tissue cells and alleviated lung injury by significantly activating SIRT3 pathway (p < 0.01). Conclusion: Theacrine alleviates inflammation and lung injury in septic mice by mediating SIRT3, thereby making it a potential lead in the development of drugs against sepsis-related inflammation and lung injury.

SIRT3 is an NAD-dependent deacetylase mainly confined to the mitochondria and its deficiency is related to the regulation of mitochondrial function and redox homeostasis [8].Previously, it has been reported that expression of SIRT3 is reduced in LPS-induced acute lung injury and SIRT3 deficiency also aggravates LPS-induced inflammation and ROS production, making acute lung injury more serious [9].So far, studies have shown that multiple drugs target SIRT3 and significantly improve the treatment effect of sepsis.Camellia assamica var kucha is a unique plant, mainly distributed in provinces of China [10].Theacrine (1,3,7,9-tetramethyluric acid), one of the major alkaloids found in Camellia assamica var kucha, is a purine alkaloid associated with bioactive functions such as antidepressant, sedative, hypnotic and lipid metabolism regulation [11,12].In addition, it has anti-inflammatory and antioxidant effects as it has been shown to reduce inflammation, and oxidative stress and improve liver damage [13,14].Furthermore, studies have revealed that Theacrine activates SIRT3 [14].However, the role and mechanism of Theacrine in sepsisinduced lung injury remain unclear.This study aims to investigate the effect of Theacrine in a sepsis model and to elucidate its potential as a drug for the treatment of sepsis.

EXPERIMENTAL Animals
Male C57BL/6 mice (40) were obtained from Shanghai Laboratory Animal Center (Shanghai, China).The experiment was approved by the Ethics Committee of Southwest Medical University (approval no.Ky2018029).Animals were handled in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines [15].Mice were injected with 3 mg/kg LPS (Sigma, USA) dissolved in 50 μL PBS or only sterile PBS (control group = 10).The remaining three groups (divided into ten mice per group) were administered theacrine (10, 20, or 40 mg/kg), (purchased from Sigma) by oral gavage 1 hour after LPS treatment.

Histological analysis
The lungs of mice were recovered after sacrificing the mice.Lung tissues were fixed with 5 % PFA, then embedded with paraffin and cut into slices.The sections were counterstained with hematoxylin-eosin staining (H&E).Lung injury score was determined by considering hemorrhage and alveolar wall thickness.Staining degree was quantified as follows: 0, 1, 2, 3 and 4 representing no staining, mild staining, moderate staining, severe staining and very severe staining, respectively.

Quantitative polymerase chain reaction (qPCR)
Total RNA was isolated from lung tissues using RNA purification Kit (Tiangen, Beijing, China) and reverse transcribed to generate cDNA according to the manufacturer's specifications.The primers are listed in Table 1.

Enzyme-linked immunosorbent assay (ELISA)
The IL-1β, TNF-α, IL-6, MDA, and SOD levels were assayed by ELISA kit (Abcam, UK).Samples were aspirated into wells and biotinconjugated primary antibodies (1:1000) were added into the wells before the addition of avidinconjugated HRP for 2 hours at room temperature.Then, enzyme substrate was added for color development and the result was measured with a microplate reader (Varioskan LUX, Thermo, USA) at 450 nm wavelength.

Immunofluorescence
Lung tissues were fixed with 5 % PFA.Afterward, sections were sliced and incubated with the ROS kit or TUNEL kit (Beyotime, Beijing, China).After rinsing with PBS and counterstaining with DAPI, the images were captured under a fluorescence microscope (Carl Zeiss, Germany).

Statistical analysis
Statistical analysis was performed using GraphPad Prism software.Data are presented as mean ± standard deviation (SD).P < 0.05 was considered statistically significant.

Theacrine relieves lung injury induced by LPS
The

Theacrine inhibits inflammatory cytokines
The effect of theacrine on inflammatory markers in LPS-induced mice is shown in Figure 2. Administration of LPS significantly increased the levels of IL-6, IL-1β and TNF-α levels (p < 0.05).However, theacrine significantly reversed the increased levels of these cytokines in a dose-dependent manner (p < 0.05; Figure 2 A).Furthermore, this trend was confirmed by the results of ELISA (Figure 2 B).Therefore, theacrine inhibits the production of inflammatory cytokines in LPS-induced mice.

Theacrine alleviates LPS-induced oxidative stress
The effect of Theacrine on LPS-induced oxidative stress was explored in the mice model.The levels of reactive oxygen species were determined by immunostaining experiment.LPS treatment increased the ROS levels in the lung tissues of mice (Figure 3 A).However, Theacrine treatment at a concentration of 10, 20 and 40 mg/kg reversed the increased intensity of ROS caused by LPS induction in lung tissues.Furthermore, Theacrine treatment significantly reduced the LPS-induced increase in the MDA levels and significantly improved the levels of SOD (p < 0.05; Figure 3 B).Therefore, Theacrine alleviates LPS-induced oxidative stress in lung tissues.

Theacrine improved LPS-induced apoptosis of lung tissues
The results of TUNEL assays revealed that LPS treatment induced apoptosis in lung tissues (Figure 4 [20].In this study, an LPS-induced sepsis mice model was recruited and used to induce lung injury.Theacrine was found to improve lung injury, oxidative stress and apoptosis caused by sepsis.
Studies have shown that damage to lung tissue caused by oxidative stress plays an important role in sepsis-induced ALI.Many factors can induce the body to produce free radicals and lead to the homeostasis disorder, so that free radicals cannot be smoothly removed, resulting in lung tissue damage and ultimately leading to ALI [19].Therefore, avoiding oxidative stress in ALI is crucial to relieving symptoms [19].In this study, Theacrine was shown to also alleviate sepsis-induced ALI via the suppression of oxidative stress.
Previously, it was shown that SIRT3 expression is reduced in LPS-induced ALI and that SIRT3 deficiency aggravates LPS-induced inflammation and ROS production, making ALI more serious [9].Therefore, SIRT3 could serve as a target for LPS-induced ALI.Interestingly, the result of this study shows that theacrine activates SIRT3 and improves LPS-induced lung tissue injury.However, the precise mechanism needs further study.

CONCLUSION
Theacrine activates SIRT3 pathway and improves LPS-induced lung injury, inflammation, oxidative stress and apoptosis in sepsis.
Therefore, it can potentially be developed as a drug for the treatment of sepsis and related inflammatory responses.
results of the histological examination of LPS-induced acute lung injury and relief score of theacrine administration are displayed in Figure 1.The mice displayed tissue injury characterized by irregular alveolar integrity.This phenotype was reversed after the administration of theacrine at a concentration of 10, 20 and 40 mg/kg.Administration of 20 and 40 mg/kg of theacrine significantly improved the lung score (p < 0.05).

Figure 2 :
Figure 2: Theacrine inhibited inflammatory cytokines.(A) mRNA levels of IL-6, IL-1β and TNF-α in lung tissues of control, LPS and theacrine-treated LPSinduced mice at the indicated concentrations of theacrine; (B) Levels of IL-6, IL-1β and TNF-α in lung tissues of control, LPS and theacrine-treated LPSinduced mice at the indicated concentration of theacrine.***P < 0.001, LPS vs control; $ p < 0.05, $$$ p < 0.001, LPS + theacrine vs LPS A).However, Theacrine suppressed apoptosis of lung tissues caused by LPS treatment (Figure 4 A).In addition, LPS treatment increased Bax as well as cleaved caspase-3 expression in the lung tissues, whereas Theacrine treatment suppressed apoptosis of lung tissues, with the decreased expression of Bax as well as cleaved caspase-3 (Figure 4 B).Therefore, Theacrine improved LPS-induced apoptosis of lung tissues.

Figure 5 :
Figure 5: Theacrine alleviated lung injury by activating SIRT3 pathway.(A) Expression levels of SIRT3 in lung tissues; (B) Levels of IL-6, IL-1β and TNF-α in lung tissues; (C) MDA and SOD levels in lung tissues; (D).Expression levels of Bax and cleaved caspase 3 in lung tissues.***p < 0.001, LPS vs control; $$$ p < 0.001, LPS + theacrine vs LPS DISCUSSION Sepsis is a clinical process of systemic inflammation caused by bacterial infection.It is a common complication in critically ill patients after surgery and is the basis for multiple organ improvement disorder syndrome.The lung is the most affected organ in multiple organ injury complicated by sepsis.Acute lung injury (ALI) appears earliest and has the highest incidence

Table 1 :
Primers used for qPCR The lung is the most affected organ in multiple organ injury complicated by sepsis.Acute lung injury (ALI) appears earliest and has the highest incidence