Involvement of kindlin‐2 in irisin’s protection against ischaemia reperfusion‐induced liver injury in high‐fat diet‐fed mice

Abstract Liver steatosis is associated with increased ischaemia reperfusion (I/R) injury. Our previous studies have shown that irisin, an exercise‐induced hormone, mitigates I/R injury via binding to αVβ5 integrin. However, the effect of irisin on I/R injury in steatotic liver remains unknown. Kindlin‐2 directly interacts with β integrin. We therefore suggest that irisin protects against I/R injury in steatotic liver via a kindlin‐2 dependent mechanism. To study this, hepatic steatosis was induced in male adult mice by feeding them with a 60% high‐fat diet (HFD). At 12 weeks after HFD feeding, the mice were subjected to liver ischaemia by occluding partial (70%) hepatic arterial/portal venous blood for 60 minutes, which was followed by 24 hours reperfusion. Our results showed HFD exaggerated I/R‐induced liver injury. Irisin (250 μg/kg) administration at the beginning of reperfusion attenuated liver injury, improved mitochondrial function, and reduced oxidative and endoplasmic reticulum stress in HFD‐fed mice. However, kindlin‐2 inhibition by RNAi eliminated irisin's direct effects on cultured hepatocytes. In conclusion, irisin attenuates I/R injury in steatotic liver via a kindlin‐2 dependent mechanism.

Kindlin-2 is a focal adhesion protein that regulates integrin signalling and cell-matrix adhesion. 19 It directly interacts with the cytoplasmic tail of β integrin, 20 which is recognized as a part of the irisin receptor. 21 A recent study has shown kindlin-2 expression is up-regulated in human and mouse fibrotic livers and depletion of kindlin-2 reduces CCL4-induced liver injury in mice. However, the role of kindlin-2 in irisin's biological function is currently unclear.
We therefore suggested that irisin attenuates hepatic I/R injury via a kindlin-2 dependent mechanism in steatotic liver. The aim of the present study was to explore the effects and likely mechanisms of irisin on hepatic I/R injury in high-fat diet (HFD)-fed mice.

| Mouse model of hepatic I/R and experimental design
After 12 weeks on a control diet or high-fat diet, the mouse hepatic I/R model was established as we described before. 17 Briefly, mice were anaesthetized with isoflurane inhalation and maintained at a concentration of 1.5%-2%. Liver ischaemia was induced by occluding partial (70%) hepatic arterial/portal venous blood for 60 minutes by a microvascular clip. Then, clip was removed and reperfusion began. Sham operation underwent all the procedures except hepatic ischaemia. There were five groups involved in present study: (a) CD-Sham: CD-fed mice underwent sham operation, and 0.5 mL saline was administrated intraperitoneally; (b) CD-I/R: CD-fed mice underwent hepatic I/R, and 0.5 mL saline was administrated intraperitoneally immediately after the initiation of reperfusion; (c) HFD-Sham: also showed as sham group, HFD-fed mice underwent sham operation and 0.5 mL saline was administrated intraperitoneally; (d) HFD-I/R: also showed as vehicle group, HFD-fed mice underwent hepatic I/R, and 0.5 mL saline was administrated intraperitoneally immediately after the initiation of reperfusion; (e) HFD-irisin: also showed as irisin group, HFD-fed mice underwent hepatic I/R, and irisin (250 μg/kg, 0.5 mL; 067-29A, Phoenix Pharmaceuticals, Inc) 17 was administrated intraperitoneally immediately after the initiation of reperfusion.

| Haematoxylin and eosin (H&E) staining and oil red O staining
The liver sections fixed in 4% paraformaldehyde were embedded in paraffin. Then cut the paraffin blocks into 5 mm-slices and stained with haematoxylin and Eosin. Liver injury score was evaluated as we described before. 17 The frozen liver sections were stained with Oil Red O to evaluate hepatic fat content.

| Western blot analysis
The protein extraction and Western blot analysis were performed as previous described. 22 The antibody information was as following:

| Immunohistochemistry and immunofluorescence staining
Immunohistochemistry and immunofluorescence staining were performed as previous described. 17 The MPO antibody (Santa Cruz

| Transfection of small interfering RNA (siRNA)
The siRNA was constructed by GenePharma Corporation and trans- (GenePharma) was used as negative control in HL-7702 cells.

| Statistical analysis
The data were expressed as mean ± standard error (SE). The t test or one-way ANOVA was used to analyse the differences between groups. SPSS version 18.0 (IBM) was used for statistical analysis, and P value < .05 was considered statistically significant.

| HFD exaggerates I/R-induced liver injury
To study the effects of HFD on hepatic I/R injury, mice were fed either a control diet (CD) or a high-fat diet (HFD) for 12 weeks. As showed in Figure S1A, HFD-fed mice were heavier than CD-fed mice (P < .05).
HFD-fed mice were associated with more prominent liver histological damage and more fat content (Figures S1B-D, P < .05). Then, HFD-fed and CD-fed mice underwent hepatic I/R (CD-I/R or HFD-I/R) or sham operation (CD-Sham or HFD-Sham). Figure S2A showed that hepatic I/R increased serum irisin level in HFD-fed mice (P < .05). As showed in Figure 1A,B, HFD induced significant liver injury characterized by hepatic steatosis (P < .05). After I/R, HFD-fed mice showed more severe liver injury and larger necrosis area than CD-fed mice ( Figure 1A-C, P < .05). The changes of serum AST and ALT were consistent with histological lesions ( Figure 1D,E, P < .05). In addition, HFD exaggerated I/R-induced ROS production ( Figure 1F,G, P < .05).

| Irisin attenuates hepatic I/R injury in HFDfed mice
Next, the effect of irisin on hepatic I/R injury in HFD-fed mice was explored. Figure 2A,B demonstrated that irisin administration reduced I/R-induced hepatic necrosis in HFD-fed mice (P < .05). Similarly, serum ALT levels were also significantly decreased by irisin treatment (118.6 ± 17.0 U/L vs 76.1 ± 6.2 U/L, Figure 2C, P < .05). Figure

| Irisin alleviates oxidative stress after hepatic I/R in HFD-fed mice
Then, the effects of irisin on oxidative stress were evaluated. As shown by liver DHE staining in Figure 3A,B, irisin administration inhibited the production of ROS compared to vehicle-treated mice (P < .05). Antioxidant GSH-Px decreased in hepatic I/R and increased after irisin administration in HFD-fed mice ( Figure 3C, P < .05).  Figure 4D-F, P < .05). As showed in Figure 4G-K, the expressions of liver endoplasmic reticulum (ER) stress-related proteins (GRP78, CHOP, PDI and Ero1-Lα) were significantly up-regulated after hepatic I/R in HFD-fed mice but reduced after irisin administration (P < .05).

| Irisin inhibits inflammatory response after hepatic I/R in HFD-fed mice
Inflammatory response was evaluated after hepatic I/R in HFD-fed mice. The recruitment of neutrophils and macrophages was detected by liver MPO and CD11b immunostaining, while the release of inflammatory factors was detected by q-PCR. In Figure 5A- Consistently, compared to HFD-fed mice of hepatic I/R, the mRNA levels of liver inflammatory factors (IL-1β, IL-6, MCP-1 and CXCL-1) were also reduced after irisin administration in HFD-fed mice ( Figure 5E-H, P < .05).

| Kindlin-2 inhibition by RNAi eliminates irisin's direct effects on cultured hepatocytes
To investigate the role of kindlin-2 in irisin's biological function, we knocked down kindlin-2 expression in HL-7702 cells ( Figure S3A,B, P < .05). The cells were exposed to palmitic acid (PA, 0.2 mmol/L) and oleic acid (OA, 0.1 mmol/L) to induce lipotoxicity injury and then subjected to hypoxia and reoxygenation treatment with/without the presence of irisin. As shown in Figure 6

| DISCUSS IONS
In the present study, using an established model of hepatic I/R in HFD-fed mice, we found that HFD exaggerated I/R-induced liver injury and ROS production. And irisin administration attenuated hepatic injury, improved mitochondrial function, and reduced oxidative and ER stress in HFD-fed hepatic I/R mice. However, in cultured hepatocytes, inhibition of kindlin-2 by RNAi eliminated irisin's effects on apoptosis, mitochondrial function, oxidative and ER stress ( Figure 7).
Hepatic I/R is a life-threaten complication in liver surgery and associated with significant morbidity and mortality, especially in hepatic I/R of steatotic liver. 6,[23][24][25][26][27] Research has shown that about 20% of patients undergoing hepatectomy have various degrees of hepatic steatosis. 28 Moreover, steatotic liver (about 20%-30% of liver donors) has been introduced as the most common type of 'extended criteria' organs due to organ shortage. [29][30][31] However, hepatic steatosis exaggerates I/R-induced liver injury, which has been proven in clinical and experimental studies. 6,[23][24][25][26][27] Although the exact mechanism remains unclear, ROS is believed to play an important role in both hepatic steatosis 32,33 and I/R injury. [34][35][36] In NAFLD, fat-laden hepatocytes are damaged by chronic oxidative/nitrosative stress (ONS). And ONS is acutely exacerbated during hepatic I/R, leading F I G U R E 3 Irisin alleviates oxidative stress after hepatic I/R in HFD-fed mice. Hepatic ischaemia was induced by occluding partial (70%) hepatic arterial/portal venous blood for 60 min, followed by 24 h of reperfusion. Sham mice underwent all the procedures except hepatic ischaemia. After 12 wk of high-fat diet (HFD), HFD-fed mice underwent sham operation (Sham) or hepatic I/R treated with 0.5 mL saline (Vehicle) or irisin (250 μg/kg, 0.5 mL). A, Liver DHE staining (red) and counterstained with DAPI (blue). Original magnification, 200×. B, The quantitative analysis of liver DHE fluorescence intensity. C, The level of liver GSH-Px. Results are expressed as mean ± SE (n = 4-5/ group) and compared by t test or one-way ANOVA. *P < .05 vs sham group, # P < .05 vs vehicle group to extensive parenchymal damage. 7 In the present study, we also showed more severe liver injury and ROS production after hepatic I/R in HFD-fed mice, suggesting inhibition of ROS production may be an effective therapeutic target.
Irisin, an exercise-induced hormone, has emerged as a key regular of energy homeostasis in obesity, diabetes and NAFLD. [37][38][39][40] A recent study has shown that irisin expression increased in non-parenchymal cells of fatty liver and was associated with the increase in innate immune cells (ie CD11b positive cells). 41 In the present study, liver CD11b positive cells were increased significantly in HFD-hepatic I/R mice, which may be the reason for the increase of serum irisin levels under such conditions. We and others have shown irisin plays a protective role in I/R of multiple organs and improvement of mitochondrial function and oxidative stress are the most common mechanisms. [9][10][11][12][13][14][15][16][17] However, the effect of irisin on I/R in steatotic liver remained unknown. The present study is the first one to reveal that irisin attenuated liver injury, improved mitochondrial function, and reduced oxidative and ER stress after I/R in steatotic liver. The dosage of irisin was based on our previous study. 17 As shown in the present study, it was also protective in HFD-hepatic I/R mice. However, the optimal dosage and the dose-dependent effect of irisin in HFD-hepatic I/R warrants further investigation. Hepatic I/R is accompanied by increased production of ROS. Mitochondria are a main source of ROS and ROS impairs mitochondrial function. [42][43][44][45] Endoplasmic reticulum (ER) stress is closely related to mitochondrial dysfunction. ER stress F I G U R E 4 Irisin improves mitochondrial function and reduces ER stress after hepatic I/R in HFD-fed mice. Hepatic ischaemia was induced by occluding partial (70%) hepatic arterial/portal venous blood for 60 min, followed by 24 h of reperfusion. Sham mice underwent all the procedures except hepatic ischaemia. After 12 wk of high-fat diet (HFD), HFD-fed mice underwent sham operation (Sham) or hepatic I/R treated with 0.5 mL saline (Vehicle) or irisin (250 μg/kg, 0.5 mL). Western blot analysis of Drp-1 (A) and its quantitative analysis (B). C, Liver relative mRNA level of Fis-1. Western blot analysis of Tfam (D) and its quantitative analysis (E). F, Liver relative mRNA level of PGC-1α. Western blot analysis of ER stress-related proteins (G) and their quantitative analysis of liver GRP78 (H), CHOP (I), PDI (J) and Ero1-Lα (K). Results are expressed as mean ± SE (n = 4-5/group) and compared by t test or one-way ANOVA. *P < .05 vs sham group, # P < .05 vs vehicle group inhibition protects steatotic and non-steatotic liver from hepatic I/R. 46 injury via binding to αVβ5 integrin. 18 However, the effect of irisin on kindlin-2, an important regulator of αVβ5 integrin function, remained unknown. In the present study, we found irisin did not change the expression of kindlin-2 after hepatic I/R in HFD-fed mice. However, kindlin-2 knockdown by RNAi eliminated the beneficial effects of irisin in hypoxia/reoxygenation-treated hepatocytes, suggesting kindlin-2 is involved in irisin's biological function.
However, whether depletion of Kindlin-2 inhibits irisin induced protection in hepatic IR in the HFD-mice warrants further investigation. And the detailed mechanism of kindlin-2 after hepatic I/R in the HFD-mice will be further explored in our future studies.
In summary, using a model of hepatic I/R in HFD-fed mice, we demonstrated that irisin attenuates I/R injury in steatotic liver. The protective effect of irisin under such conditions requires kindlin-2.
Irisin may be a novel effective treatment for NAFLD patients with hepatic I/R.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Rongqian Wu
https://orcid.org/0000-0003-0993-4531 F I G U R E 7 Irisin attenuates hepatic I/R injury via a kindlin-2 dependent mechanism in steatotic liver. HFD exaggerated I/R-induced liver injury and ROS production. And irisin administration attenuated liver injury, improved mitochondrial function, and reduced oxidative and ER stress in HFD-fed hepatic I/R mice. However, inhibition of kindlin-2 by RNAi eliminated irisin's effects on apoptosis, mitochondrial function, oxidative and ER stress in cultured hepatocytes