The protective effect of PPARγ in sepsis-induced acute lung injury via inhibiting PTEN/β-catenin pathway

The present study aims to reveal the molecular mechanism of peroxisome proliferator-activated receptor γ (PPARγ) on sepsis-induced acute lung injury (ALI). To do that, the rat injury model was established using cecal ligation and perforation (CLP) method, followed by different treatments, and the rats were divided into Sham group, CLP group, CLP + rosiglitazone (PPARγ agonist) group, CLP + GW9662 (PPARγ inhibitor) group, CLP + bpV (phosphatase and tensin homolog (PTEN) inhibitor) group, CLP + GW9662 + bpV group. Compared with Sham group, the mRNA and protein expression levels of PPARγ were down-regulated, the inflammation levels were elevated, and the apoptosis was increased in CLP group. After treatment with rosiglitazone, the protein expression level of PPARγ was significantly up-regulated, the phosphorylation level of PTEN/β-catenin pathway was decreased, the PTEN/β-catenin pathway was inhibited, the lung injury, inflammation and apoptosis were reduced. The opposite effect was observed after treatment with GW9662. Besides, bpV inhibited PTEN/β-catenin pathway, and relieved the lung tissue injury. The overexpression of PPARγ reduced inflammatory response and inhibited apoptosis in sepsis-induced ALI. Furthermore, PPARγ relieved the sepsis-induced ALI by inhibiting the PTEN/β-catenin pathway.


Introduction
Sepsis is an organic disease induced by abnormal host reaction to infection [1]. Besides, sepsis-induced acute lung injury (ALI) is proved to commonly lead a higher mortality rate than other causes of ALI [2,3]. Although various therapy strategies have been successfully used for clinical treatment of sepsis-induced ALI, the efficacy of these strategies is still not ideal [4]. Thus, a deep understanding of the molecular mechanism of the progression of sepsis-induced ALI is beneficial for effective clinical therapies.
The relationship between peroxisome proliferator-activated receptor γ (PPARγ) and ALI has been proved by previous studies [5,6]. The mRNA expression of PPARγ in lung tissues is decreased in ALI mice, and keeps at a low level at the end of the observation period [7]. The increased expression of PPARγ is critical to protect against ALI in mice [8]. In addition, PPARγ also plays a key regulatory role in acute sepsis and sepsis-induced immunosuppression [9]. Brenneis et al. have indicated that the expression of PPARγ in T cells can be used as a prognostic marker of sepsis [10]. Rosiglitazone is a well-known antidiabetic oral drug which binds to PPAR, allowing the cells to be responsive to insulin [11]. As an agonist of PPARγ, rosiglitazone significantly suppresses LPS-induced ALI in mice [12]. Actually, the biological function of PPARγ in disease progression is commonly realized by targeting certain genes or pathways such as phosphatase and tensin homolog (PTEN) and PTEN/β-catenin pathway [13,14]. The PTEN/β-catenin signaling pathway is closed related to the inflammatory responses in liver and reperfusion injuries [15]. Although previous studies have mentioned the biological function of miR-PPARγ and its related genes or pathways in sepsis or ALI, the detailed molecular mechanism of PPARγ in the progression of sepsis-induced ALI is still unclear.
In the present study, the sepsis-induced ALI rat model was established via cecal ligation and puncture (CLP). An agonist of PPARγ, rosiglitazone was used to up-regulate PPARγ, and an inhibitor of PPARγ, GW9662 was used to down-regulate PPARγ. The effects of PPARγ were then analyzed on lung tissues and cells in sepsis-induced ALI rats. Based on that, we further explored the molecular mechanism of PPARγ involving PTEN/β-catenin pathway in sepsis-induced ALI.

Establishment of ALI model
A total of 70 male Sprague-Dawley (SD) rats (320-370 g, 6-8 months) were obtained from Animal Laboratory Center of General Hospital of Nanjing Military Region. Rats were housed under standard conditions (22 • C, 50% relative humidity, 12-h/12-h light/dark cycle) with free access to water and food. All rats were divided into blank control group (blank group, n=10), sham operated group (Sham group, n=10), model group (CLP group, n=10), CLP + Rosiglitazone group (n=10), CLP + GW9662 group (n=10), CLP + bpV group (n=10) and CLP + GW9662 + bpV group (n=10). At the beginning of operation, a total of 150 mg/kg of imidazole sodium (analgene, 500 mg/ml, Sanofi-Aventis) was intraperitoneally injected into rats to prevent postoperative pain. Briefly, the anesthesia with sodium pentobarbital (50 mg/kg) was performed on rats via intraperitoneal injection. A 2-cm incision was made along the midline of the abdomen. The root of the cecum was ligated annularly with 4-0 silk thread. Then, the feces were squeezed out with 18G needle at the free end once and sent back to the abdomen. Finally, the peritoneum and skin were sutured in turn. In Sham group, only laparotomy, distal cecum separation and abdominal closure were performed. The CLP + rosiglitazone group was intraperitoneally injected with 5 mg/kg rosiglitazone (R2408, Sigma-Aldrich). The CLP + GW9662 group was intraperitoneally injected with 5 mg/kg GW9662 (M6191, Sigma-Aldrich). The CLP + bpV group was intraperitoneally injected with 200 nmol/kg bpV (bpV(phen), sc-221378, Santa Cruz Biotechnology). CLP + GW9662 + bpV group was intraperitoneally injected with 5 mg/kg GW9662 and 200 nmol/kg bpV. The doses of the above agents were determined by our preliminary experiments in accordance with previous studies [16][17][18]. The above agents were all injected at 30 min before CLP (the efficacy could be fully reflected at this time point) in accordance with our preliminary experiments and previous studies [16,19]. The present study was approved by the ethics committee of Qilu Hospital of Shandong University, and all experiments were in accordance with the local guide for the care and use of laboratory animals. All animal experiments were performed at the Department of Lab Animal Science of Qilu Hospital of Shandong University.

Lung wet-to-dry weight ratios measurement
All rats were killed by dislocation 24 h after modeling, and before that, the mortality was 0, followed by obtaining the right lower lung lobes of each rat. Briefly, the wet-to-dry weight ratio was calculated according to the wet weight (W) and dry weight (D) of the lung tissue. Meanwhile, the fresh lung tissue blocks were fully shredded, poured into a glass homogenate tube and thoroughly ground. The prepared 5% tissue homogenate was centrifuged at 3000 rpm for 10 min, and the supernatant was stored for further use. The activity for myeloperoxidase (MPO) assay was performed according to the instructions of the myeloperoxidase test kit (Nanjing Jian Bioengineering Co., Ltd.). Then, the absorbance at 460 nm was measured. The results are expressed as unit of enzyme activity.

Bronchoalveolar lavage assay and cell counting
Bronchoalveolar lavage fluid (BALF) was obtained by tracheal intubation, injection and recovery of sterile PBS. BALF was centrifuged for 10 min at 4 • C for 2000 rpm. The total number of cells was measured by Hematology analyzer (Abbott CELL-DYN 4000). BALF cell smears were stained with Giemsa, and the neutrophils and macrophages were counted.

ELISA
The lung tissue (100 mg) was ground with a mortar and pestle in PBS, and the supernatant was taken after centrifugation. The expression levels of TNF-α/IL-1β/IL-6 in BALF were determined by the ELISA kit (Biosource International, Inc., California, U.S.A.). The standard curve of optical density (OD) value and concentration were drawn, and the concentration of the sample was calculated according to the standard curve.

HE staining investigation
Lung tissues of the rats in each group were fixed with 10% neutral formaldehyde solution for 24 h, followed by routine dehydration, transparency, paraffin immersion, embedding and coronal section (4 μm). The sections were dewaxed with xylene, dehydrated with ethanol of different gradient concentrations, and then stained with Hematoxylin. The pathological changes were observed under a light microscope (Olympus, Japan). The lung injury score (0-4) was calculated by two independent pathologists.

TUNEL assay
The TUNEL assay was performed to detect the apoptosis from each group. According to the instructions of the TUNEL kit (Ruisai Biotechnology Co., Ltd., Shanghai, China), the lung tissue (1 cm × 1 cm × 0.5 cm) was fixed with 4% formaldehyde, conventional dewaxed with xylene, rehydrated with gradient alcohol and water, and then placed in PBS for 5-10 min. Next, the cells were cultured with the mixture of TUNEL reaction at 37 • C for 1 h. Finally, the apoptosis was observed by FV300 microscope (Olympus).

The qRT-PCR assay
The total RNAs from sample of each group were extracted and quantified using TRIzol™ Plus RNA kit (Invitrogen, Carlsbad, CA, U.S.A.), and the cDNA was synthesized by SuperScript™ IV First-Strand Synthesis System kit (Invitrogen, Carlsbad, CA, U.S.A.). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference. The qRT-PCR was performed with ABI7500 quantitative PCR instrument. The reverse transcription primers were obtained from reverse transcription kit (TaKaRa). The primers were shown in Table 1. The reaction conditions were as follows: 95 • C for 3 min, 39 cycles at 95 • C for 10 s, 55 • C. The threshold value was selected manually as the lowest point of parallel rise of logarithmic amplification curves, followed by obtaining the threshold cycle (C t ) value. Finally, the relative expression of candidate genes was calculated by 2 − C T method [20].

Western blot
Lung tissues from each group were lysed with 100 μl/50 ml protein lysate RIPA. Briefly, the extracted protein was quantified by bicinchoninic acid (BCA) method. A total of 10% polyacrylamide gels were used for the separation of proteins. Then, the gels were transferred to polyvinylidenefluoride membranes. After blocking with 5% Skim milk/BSA, the membrane was incubated with primary antibodies (

Statistical analysis
Data were analyzed using SPSS 22.0 (Chicago, IL, U.S.A.) and GraphPad Prism 7.0. One-Way ANOVA was used for the current study. Tukey's multiple comparison test was used for the pairwise comparison after ANOVA. P<0.05 was considered as statistically significant. All trials were repeated at least three times.

Down-regulation of PPARγ led to the poor survival of ALI rats
The qRT-PCR and Western blot showed that there were no significant differences in the mRNA and protein expression of PPARγ between the Blank and the Sham groups (all P>0.05). Compared with the Sham group, the mRNA and protein expression of PPARγ in the CLP group were significantly decreased (all P<0.01). Compared with the CLP group, the expression of PPARγ was significantly up-regulated and down-regulated by PPARγ agonist rosiglitazone and PPARγ inhibitor GW9662, respectively (all P<0.05) ( Figure 1A,B). In addition, the survival rate of rats in the CLP group was lower than that in the Sham group at 24 h post-CLP (P<0.05). The intervention of rosiglitazone and GW9662 significantly prolonged and shortened the survival rate of ALI rats underwent CLP, respectively (P<0.05) ( Figure 1C).

PPARγ inhibited cell apoptosis and alleviates lung injury in rats
The result of HE staining showed that the alveolar structure of blank and Sham groups was clear and complete. Meanwhile, the alveolar wall in most areas of CLP group was markedly edema and widened, and a large number of inflammatory cell infiltration and erythrocyte exudation were observed in alveolar wall and alveolar cavity. The lung injury score was significantly higher in the CLP group than that in the Sham group (P<0.01). Compared with the CLP group, the pathological changes were relieved and enhanced by rosiglitazone and GW9662, respectively ( Figure 2A). The intervention of rosiglitazone and GW9662 significantly decreased and increased the lung injury score, respectively (P<0.01) ( Figure 2B). Furthermore, TUNEL assay showed that there were only a few apoptotic

PPARγ inhibited pulmonary inflammation and alleviates lung injury in CLP rats
The ratio of wet to dry weight of lung tissues showed that compared with Sham group, the ratio in CLP group was significantly increased (P<0.01) ( Figure 3A). Compared with CLP group, the ratios in CLP + rosiglitazone group and CLP + GW9662 group were significantly lower and higher, respectively (all P<0.05). The results of MPO activity test in lung tissue showed that there was no significant difference in MPO activity between blank group and Sham group ( Figure 3B). Compared with Sham group, MPO activity in CLP group was increased significantly (P<0.01). Compared with CLP group, MPO activity in CLP + rosiglitazone group and CLP + GW9662 group was significantly decreased and increased, respectively (all P<0.05). The level of inflammation in BALF was detected by ELISA ( Figure 3C). Compared with Sham group, the levels of TNF-α, IL-1β and IL-6 in CLP group were significantly higher (P<0.01). Compared with CLP group, the levels of TNF-α, IL-1β and IL-6 in CLP + rosiglitazone group were significantly lower (P<0.05), while the levels of inflammation in CLP + GW9662 group were significantly higher (P<0.05). Giemsa staining showed that compared with Sham group, the total number of BALF cells, neutrophils and macrophages in CLP group were increased significantly (all P<0.05) ( Figure 3D). Compared with CLP group, the total number of BALF cells, neutrophils and macrophages in CLP + rosiglitazone group and CLP + GW9662 group were significantly decreased and increased, respectively (all P<0.05).

Down-regulation of PPARγ activated PTEN/β-catenin pathway in CLP rats
To explore the protective effect of PPARγ on lung injury in CLP rats through PTEN/β-catenin pathway, the expression and phosphorylation level of PETN/β-catenin pathway in CLP rats were detected by intraperitoneal injection of PTEN/β-catenin pathway inhibitor bpV before CLP (Figure 4). Compared with CLP group, the phosphorylation levels of PTEN and β-catenin were decreased significantly, while the phosphorylation levels of Akt and GSK-3β were increased significantly in CLP + bpV group (all P<0.01). Meanwhile, compared with CLP group, the phosphorylation levels of PTEN and β-catenin were increased significantly, while the phosphorylation levels of Akt and GSK-3β were decreased significantly in CLP + GW9662 group (all P<0.01). Compared with CLP + GW9662 group, the phosphorylation levels of PTEN and β-catenin in CLP + GW9662 + bpV group were decreased significantly, while the phosphorylation levels of Akt and GSK-3β were increased significantly (all P<0.01).

The bpV reversed the effect of GW9662 on apoptosis and lung injury
HE staining was used to detect the effect of bpV on lung injury. Compared with the CLP group, the lung injury score in the CLP + GW9662 group was significantly increased (P<0.01). Compared with the CLP + GW9662 group, the lung injury score in the CLP + GW9662 + bpV group was significantly reduced (P<0.05) ( Figure 5A,B). TUNEL was used to detect apoptosis. Compared with the CLP group, the apoptosis in the CLP + bpV group and the CLP + GW9662 group was significantly reduced and increased, respectively (all P<0.01). Compared with CLP + GW9662 group, the apoptosis of CLP + GW9662 + bpV group was significantly reduced (P<0.01) ( Figure 5C,D).

The bpV reversed effect of GW9662 on pulmonary edema and inflammatory response in CLP rats
Compared with the CLP group, the wet to dry weight ratio ( Figure 6A), MPO activity ( Figure 6B), TNF-α/IL-1β/IL-6 levels ( Figure 6C), and the total cells, neutrophils and macrophages in the BALF ( Figure 6D) were significantly decreased in the CLP + bpV group (all P<0.01). Compared with the CLP group, the above parameters in the CLP +

Figure 4. Bpv blocked PTEN/β-catenin pathway
The phosphorylation of PTEN/β-catenin pathway was detected by Western blot. The y-axis represented the relative protein expression of phosphorylated PTEN, total PTEN, phosphorylated AKT, total AKT, phosphorylated GSK-3β, total GSK-3β, phosphorylated β-catenin, and total β-catenin; the x-axis represented different protein expression in CLP group, CLP+GW9662 group, CLP+bpv group and CLP+GW9662+bpv group, respectively. The expression of protein was quantified by normalizing to GAPDH and relative to the CLP group (set as 1). Tukey's multiple comparison test followed by One-Way ANOVA was used for the pairwise comparison. ## , P<0.01 compared with CLP + GW9662 group; **, P<0.01 compared with CLP group. GW9662 group were significantly increased (all P<0.01). Moreover, compared with the CLP + GW9662 group, the above parameters in the CLP + GW9662 + bpV group were significantly reduced (all P<0.01).

Discussion
Inflammation is one of the outstanding characteristics during the development of sepsis-induced ALI [21]. Based on the rat model, reduced inflammation is the optimal target of molecular therapy for sepsis-induced ALI [22]. A previous study showed that the inflammation can lead to a rapid and profound promotion in lung vascular permeability [23]. The development of clinical drugs for sepsis-induced ALI has been focused on the anti-inflammation during the process of this disease [24]. Despite of inflammation, the increasing apoptosis has also been proved to be one of the most common characteristics in sepsis-induced ALI [25]. Canikli et al. showed that the apoptosis was significantly increased in epithelial cell of sepsis-induced ALI [26]. Actually, some biological molecules play vital roles in the anti-inflammation or anti-apoptosis during the progression of disease [27]. A previous study showed that PPARγ attenuated intimal hyperplasia by inhibiting inflammation in vascular smooth muscle cells [28]. Kohno et al. indicated that the PPARγ agonist rosiglitazone reduced colonic inflammation in mice model [29]. Via the down-regulation of PPARγ, previous study showed that tissue transglutaminase activation could modulate the inflammation in cystic fibrosis [30]. Meanwhile, PPARγ controls the cell proliferation and apoptosis in the development of lung disease [31].
However, Bassaganyariera et al. indicated that PPARγ was highly expressed in adipose tissue macrophages and dampened adipose-tissue inflammation [32]. Although the anti-inflammation or anti-apoptosis effect of PPARγ has been mentioned in sepsis or ALI [8,9], but the detail biological function of PPARγ in sepsis-induced ALI is still unclear. In the present study, the expression of PPARγ in sepsis-induced ALI was significantly down-regulated, while inflammatory factors levels and apoptosis was significantly increased. Meanwhile, with the treatment of agonist rosiglitazone, the protein expression of PPARγ was significantly increased, while inflammation level and apoptosis significantly decreased. Thus, the up-regulation of PPARγ reduced inflammatory response and inhibited apoptosis in sepsis-induced ALI. Furthermore, we found that the intervention of rosiglitazone and GW9662 significantly prolonged and shortened the survival rate of sepsis-induced ALI rats, respectively. These findings further indicated that PPARγ is a novel therapeutic target for sepsis-induced ALI. PTEN is proved to play an important role during the inflammatory response of various biological processes [33]. Meanwhile, a previous study indicated that the activation of β-catenin contributed to the immune homeostasis [34]. Based on a mouse model, a previous study showed that PTEN/β-catenin signaling pathway could promote T cell induction of liver ischemia and reperfusion injury [15]. Lai et al. indicated that the down-regulation of PTEN expression protected the development of ALI in mice [35]. In the study of sepsis-induced cardiomyocyte apoptosis, Yao et al. showed that miR-25 could inhibit cell apoptosis via targeting PTEN [36]. Meanwhile, a previous study showed that the β-catenin associated pathways participated in the progression of lung injury [37]. Inhibition of β-Catenin related signaling can promote engraftment of mesenchymal stem cells to repair ALI [38]. Importantly, based on an animal model, a previous study indicated that PTEN/β-catenin signaling regulated the inflammation process during liver injury [15]. In the current study, after treatment with PPARγ agonist rosiglitazone, the phosphorylation level of PTEN/β-catenin pathway was significantly decreased, while the degree of lung injury was reduced. Interestingly, the opposite effect was observed after treatment with PPARγ inhibitor GW9662. Meanwhile, the Western blot showed that PTEN inhibitor bpV attenuated lung injury induced by GW9662. Thus, we speculated that PPARγ has a protective effect on sepsis-induced ALI by inhibiting the expression of PTEN/β-catenin signaling pathway. However, there were still some limitations in the current study including small sample size and lack of verification analysis. Thus, a further verification study based on a large sample size is needed to confirm all speculation in the present study.
In conclusion, PPARγ has a protective effect on sepsis-induced ALI by inhibiting the expression of PTEN/β-catenin signaling pathway. Furthermore, the up-regulation of PPARγ reduced inflammatory response and inhibited apoptosis, which might be a novel therapy target for sepsis-induced ALI.