p-JAK2 plays a key role in catalpol-induced protection against rat intestinal ischemia/ reperfusion injury †

Catalpol, an iridoid glucoside isolated from the radix of Rehmannia glutinosa Libosch, is found to have a wide variety of biological activities. Based on our pre-experiments, we proposed that catalpol pretreatment could protect against intestinal ischemia/reperfusion (I/R) injury and the JAK2/STAT3 signaling pathway might play an important role in the protection. Both in vivo intestinal I/R-injured rats and in vitro hypoxia/reoxygenation (H/R)-injured IEC-6 cells were used in the present study. The results con ﬁ rmed our proposal. The in vivo results showed that catalpol (25, 50 mg kg (cid:1) 1 ) signi ﬁ cantly attenuated rat intestinal I/R injury by decreasing pro-in ﬂ ammatory cytokines, reducing oxidative stress, and restoring intestinal barrier function without a ﬀ ecting the corresponding controls. Catalpol pretreatment signi ﬁ cantly increased the Bcl-2/Bax ratio, and decreased cleaved caspase3, p-JAK2, and p-STAT3 protein expression without a ﬀ ecting total JAK2 and STAT3 in vivo and in vitro , showing that catalpol inhibited apoptosis through selectively inhibiting p-JAK2 and p-STAT3. In vitro studies indicated that catalpol (40 m M) pretreatment signi ﬁ cantly and selectively inhibited p-STAT3 nuclear translocation and transfection of JAK2 siRNA signi ﬁ cantly decreased JAK2, p-JAK2, STAT3, p-STAT3, and other apoptosis-related proteins in H/R-injured IEC-6 cells, suggesting that JAK2 siRNA partially simulated the e ﬀ ects of catalpol on apoptosis-related proteins. JAK2 siRNA + catalpol could not further decrease JAK2, p-JAK2, STAT3, p-STAT3, and other apoptosis-related proteins, suggesting that inhibition of the JAK2/STAT3 signaling pathway plays a key role in catalpol-induced protective e ﬀ ects. Our results suggest that p-JAK2 is the key target in catalpol-induced protection against intestinal I/R injury, providing information for further translational studies. present was designed to verify our proposal by using rat I/R In summary, the present study demonstrated that catalpol pretreatment signi  cantly attenuated rat intestinal I/R injury by inhibiting oxidative stress and in  ammation, restoring intestinal barrier function, and decreasing mitochondria-mediated apoptosis through blocking JAK2/STAT3 signaling pathway via selective inhibition of p-JAK2. Catalpol did not signi  cantly a ﬀ ect the corresponding controls. Furthermore, catalpol is low toxicity, and has anti-bacterial activity which may inhibit bacterial infection during the development of intestinal I/R injury. Our study suggests that p-JAK2 plays a key role in catalpol-induced protection on intestinal I/R injury and p-JAK2 can be chosen as a therapeutic target, providing valuable information for the further translational studies of catalpol in protecting against

To effectively ameliorate I/R-induced tissue injuries and reveal the correlated mechanisms, natural health promoting compounds which possess diverse biological effects are believed to be potentially benecial. Rehmannia glutinosa Libosch (known as Dihuang in China) has been used for centuries as an oriental traditional dietary herb or functional food. 15,16 Catalpol, an iridoid glucoside of the main active component in the radix from Rehmannia glutinosa Libosch, 17,18 is found to have a wide variety of biological activities including anti-oxidative, anti-inammatory, anti-cancer, hypoglycemic, and analgesic effects. 19,20 Catalpol is found to have protective effects against tissue injuries including liver, kidney, and brain injuries. 19,21,22 Catalpol also shows a strong anti-microbial effect against intestinal infection caused by pathogens during intestinal I/R. 19,23 However, whether catalpol can effectively protect against intestinal I/R injury remains unknown.
Based on our pre-experiments, we proposed that catalpol pretreatment could protect against intestinal I/R injury by inhibiting I/R-induced inammation, oxidative stress, apoptosis, and intestinal barrier dysfunction, and JAK2/STAT3 signaling pathway might play an important role in catalpolinduced protective effects. The present study was designed to verify our proposal by using rat I/R injury model in vivo and using hypoxia/reoxygenation (H/R)-injured rat small intestinal crypt epithelial cells (IEC-6 cells) in vitro.

Animals
Male Sprague-Dawley (SD) rats weighting 200-220 g were obtained from the Experimental Animal Center of Dalian Medical University (Certicate of Conformity: No. SCXK 2008-2002). All rats were fed for one week before experiment in a room temperature of 22 AE 2 C with 12 h light-to-dark cycle and provided standard food and water ad libitum. The experimental protocol was carried out based on the Declaration of Helsinki and supported by Dalian Medical University Animal Care and Ethics Committee. Human subjects are not involved in the present study.

Establishment of intestinal I/R injury rat model
Before surgery, rats were fasted for 12 h with free access to water and then were anesthetized with sodium pentobarbital (50 mg kg À1 body weight, i.p.) and subjected to a 2 cm midline abdominal incision. The rat intestinal I/R model was established by superior mesenteric artery (SMA) occlusion as previously described. 24 The SMA was isolated and clamped with an atraumatic microvascular clamp for 1 h, and then, the clamps were removed to allow 2 h reperfusion. 3 Aer reperfusion, blood samples were withdrawn via abdominal aorta and small intestinal segments were prepared for analysis.
Catalpol was dissolved in normal saline (NS), and rats were administered catalpol or NS intragastrically (i.g.) once daily in a volume of 2 mL kg À1 . Rats were assigned randomly into ve groups (n ¼ 8 in each group): (1) sham group: rats were administered i.g. NS for three consecutive days and underwent isolation of SMA without occlusion; (2) catalpol (H) + sham group: rats were pretreated with catalpol at a dose of 50 mg kg À1 once daily for three consecutive days before surgery and underwent isolation of SMA without occlusion; (3) I/R group: rats were administered i.g. NS and were subjected to 1 h intestinal ischemia and 2 h reperfusion; (4) catalpol (L) + I/R group: rats were pretreated with catalpol at a dose of 25 mg kg À1 once daily for three consecutive days before surgery and were subjected to 1 h intestinal ischemia and 2 h reperfusion; (5) catalpol (H) + I/R group: rats were pretreated with catalpol at a dose of 50 mg kg À1 once daily for three consecutive days before surgery and were subjected to 1 h intestinal ischemia and 2 h reperfusion.

Cell culture and H/R
IEC-6 cells were cultured in Dulbecco's modied Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 0.1 U mL À1 bovine insulin. IEC-6 cells were maintained in a humidied atmosphere containing 5% CO 2 at 37 C. To stimulate hypoxic conditions, IEC-6 cells were incubated in a microaerophilic system with 5% CO 2 and 1% O 2 and balanced with 94% N 2 for 4 h. The cells were cultured for 3 h under normoxic conditions to achieve reoxygenation.

Cell viability assay
IEC-6 cells were seeded in 96-well plates and grown overnight. The cells were treated with gradient dilution of catalpol (2.5, 5, 10, 20, 40, 80, 160 mM) and incubated for 6 h, 12 h, and 24 h at 37 C with 5% CO 2 before H/R. According to the protocol, CCK-8 regent (10 mL) was added to each well for 1 h incubation at 37 C, which was measured at 450 nm using a microplate reader (PerkinElmer, USA).

Histological analysis and in situ TUNEL staining
Aer sacrice, the isolated rat small intestinal segments were inated in 4% paraformaldehyde solution. Paraffin sections (4 mm thickness) of intestine were prepared and stained with haematoxylin and eosin (H & E). The histopathological score was selected for evaluation of lesions in intestinal tissues by using Chiu's method. Grade 0 represents normal villi and grades 1-5 indicates increments of severity of injury (grade 1: capillary congestion; grade 2: moderate liing of epithelial layer; grade 3: severe progressive liing of epithelial layer; grade 4; denuded villi, dilated capillaries; grade 5: digestion & disintegration of lamina propria, hemorrhage and ulceration). Determination of apoptosis in tissue was performed using an in situ cell death detection kit (Keygen Biotech, China) according to the manufacturer's instructions.

In vitro apoptotic index determination
Measurement of in vitro apoptotic cell death was performed using a TUNEL Apoptosis Detection Kit from Roche. IEC-6 cells were xed in 4% paraformaldehyde (PFA) for 30 min at room temperature. Then, the xed cells were incubated with 0.1% TritonX-100 for 10 min. Aer washing cells with phosphatebuffered saline (PBS), 50 mL of TUNEL reaction mixture was added, and cells were incubated at 37 C for 1 h and then observed under a uorescence microscope (Olympus, Japan).
Acridine orange/ethidium bromide (AO/EB) staining indicated that the viable cells exhibited bright green and the apoptotic cells showed orange color. IEC-6 cells were stained with AO (200 mg mL À1 ) and EB (200 mg mL À1 ) for 10 min and then washed with PBS to remove background staining. And the cells were then observed under a uorescence microscope.

Immunouorescence staining
The nuclear translocation of p-STAT3 was examined using immunouorescence staining. IEC-6 cells were cultured in chamber slides and then treated with catalpol at the concentration of 40 mM. The cells were washed with PBS and xed with 4% paraformaldehyde for 20 min at room temperature. Samples were permeabilized with 0.4% TritonX-100 for 10 min, and washed with PBS for three times followed by blocking with 2% bovine serum albumin (BSA) in PBS at 37 C for 1 h. Rabbit anti-p-STAT3 (1 : 50) in the 1% blocking solution was added into the samples and incubated overnight at 4 C. Following three 5 min washes with PBS, uorescein-conjugated secondary antibody (1 : 100) was added in 1% blocking solutions and incubated for 1 h. The stained samples were mounted with DAPI (1 mg mL À1 for 10 min) to stain cell nuclei. Aer three additional 5 min washes, the samples were examined and analyzed using a uorescence microscope.
Small intestine sections were incubated with rabbit antioccludin (1 : 50, Proteintech, Wuhan, China) or anti-ZO-1 (1 : 50, Santa Cruz, CA, USA) in a humidied chamber overnight at 4 C. Aer washing with PBS for three times, the sections were incubated with a uorescein-conjugated secondary antibody (1 : 100, Beyotime Biotechnology, Shanghai) for 1 h. Aer three additional 5 min washes, the samples were examined and analyzed using a uorescence microscope.

Biochemical analysis of intestinal tissues
Serum I-FABP and DAO activity were determined using ELISA kits according to the manufacturer's instructions. Small intestine sections of rats were homogenized in ice-cold normal saline. The homogenates were centrifuged at 4000 g min À1 at 4 C for 10 min. Then pro-inammatory cytokines and mediators, including IL-6, IL-1b, and TNF-a, were determined by using ELISA kits. The contents of MDA, SOD, GSH, and MPO in small intestine were measured by using the commercial kits according to the manufacturer's instructions.

Small RNA interference transfection
Transfection of small interfering RNA (siRNA) was used to down-regulate JAK2. IEC-6 cells were plated into 6-well plates for 24 h and then transfected with JAK2 siRNA or control siRNA (GenePharma, Shanghai, China) using Lipofectamine 2000 reagent (Invitrogen, Shanghai, China) according to the manufacturer's instructions. Six hours aer transfection, the cells were treated with catalpol before H/R injury, and then the protein levels of JAK2, p-STAT3, STAT3, Bax, Bcl-2, and cleaved caspase3 were measured.

Statistical analysis
Data are presented as the mean AE SEM. According to the GraphPad Prism 5.0 soware (GraphPad Soware Inc., San Diego, CA, USA), a two-tailed Student's t-test was used to compare means between two groups. Statistical signicance was inferred at P-values of less than 0.05.

Effects of catalpol on rat intestinal I/R injury in vivo
3.1.1. Effects of catalpol on I/R-induced intestinal morphologic injuries. Morphological signs of small intestine injuries were observed using microscopy. The protective effects of catalpol on histological changes of rat intestinal I/R injury were determined by using H & E staining. There were no observable morphologic signs of injuries in the intestinal tissues of both sham rats and catalpol (H) + sham rats. Significant intestinal morphologic alterations were observed in I/Rinjured rats, which manifested as denuded villi, dilated capillaries, digestion and disintegration of lamina propria, hemorrhage, and ulceration. I/R-induced rat intestinal injury was signicantly attenuated by catalpol (25, 50 mg kg À1 ) pretreatment (Fig. 1B). Meanwhile, the Chiu's score was signicantly increased in I/R-injured rats compared with the sham rats, and catalpol pretreatment signicantly reversed the increased the Chiu's score (Fig. 1C), showing the protective effects of catalpol on intestinal I/R injury.
3.1.2. Effects of catalpol on intestinal I/R-induced oxidative stress. MDA is a biomarker of oxidative damage, 25 and SOD and GSH are biomarkers of antioxidant capacity. 26 Compared with sham rats, the intestinal MDA level was signicantly increased, and the intestinal SOD and GSH levels were markedly decreased in I/R-injured rats. Catalpol (50 mg kg À1 ) pretreatment not only signicantly reversed the increased MDA level but also signicantly reversed the decreased SOD and GSH levels ( Fig. 2A). Catalpol did not affect those indicators in catalpol (H) + sham rats.  27 and increase of inammatory cytokines including IL-6, IL-1b, and TNF-a. 28 And I-FABP is also an important indicator of both intestinal tissue injury and inammation. 29 Our results indicated that I-FABP and other inammatory parameters including IL-6, IL-1b, TNF-a, MPO were signicantly increased in I/R-injured rats compared with sham rats. Catalpol pretreatment (25, 50 mg kg À1 ) signicantly reversed these increased inammatory indicators (Fig. 2B). Catalpol did not signicantly affect those inammation-related indicators in catalpol (H) + sham rats.

Effects of catalpol on intestinal I/R-induced inammation. Inammation induced by intestinal I/R is characterized by an increase in MPO activity (an index of neutrophil inltration),
3.1.4. Effects of catalpol on I/R injured rat intestinal barrier function. DAO is synthesized in epithelial cells of intestinal villi which are usually an epicenter of the ischemia. 30 And tight junction (TJ) proteins including occludin and ZO-1 are vital for normal intestinal architecture. 31 The disturbance of the distribution and expression of these proteins are detected in intestinal barrier injuries. In intestinal I/R-injured rats, DAO serum level signicantly increased and TJ proteins markedly reduced compared with the sham-operated rats (Fig. 3A). The ELISA result showed that catalpol pretreatment signicantly reduced serum DAO level of I/R-injured rats compared with I/Rinjured rats. According to western blot analysis and immuno-uorescence assays, the results showed that catalpol pretreatment markedly reversed the decreased expressions of both occludin and ZO-1 proteins in I/R-injured rats compared with intestinal I/R-injured rats (Fig. 3B and C). Catalpol did not signicantly affect these indicators in catalpol (H) + sham rats.
3.1.5. Effects of catalpol on intestinal I/R-induced apoptosis in vivo. Mitochondria-mediated apoptosis plays a central role in tissue homeostasis. 32 The decrease of Bcl-2/Bax ratio is found to activate the mitochondrial apoptotic pathway, and cleaved caspase3 is a mark of apoptotic execution protein. 33 JAK2 activation, following by activation of STAT3, stimulates apoptosis. 34 Apoptosis induced by intestinal I/R was detected using TUNEL staining and the expression of apoptosis-related proteins was measured using western blot analysis. Our results showed that intestinal I/R signicantly increased apoptosis, and catalpol pretreatment signicantly reversed the increased apoptosis by reducing the TUNEL-positive cells (Fig. 4A), by increasing Bcl-2/Bax ratio, and by decreasing the expression of cleaved caspase3 (Fig. 4C). Western blot analysis showed that p-JAK2 and p-STAT3 were increased in I/R-injured rats and catalpol pretreatment (25, 50 mg kg À1 ) signicantly and selectively reversed the increased p-JAK2 and p-STAT3 without affecting total JAK2 and STAT3 (Fig. 4B). Catalpol did not affect those in catalpol (H) + sham rats. The results suggested that JAK2/STAT3 pathway is involved in the protective effects of catalpol on intestinal I/R injury. cells were pretreated with gradient dilution of catalpol in normal and H/R conditions for 12 h. The CCK-8 assay showed that catalpol, at the concentration of 80 mM or lower, did not affect the viability of IEC-6 cells (Fig. 5A). Compared with the H/ R-injured control, catalpol pretreatment at concentrations of 10, 20, and 40 mM on H/R-injured IEC-6 cells for 12 h signicantly reversed the decreased cell viability (Fig. 5B and C), indicating that catalpol pretreatment selectively protected against H/R-induced cell injury.

Effects of catalpol on H/R-injured
3.2.2. Effects of catalpol on H/R-induced apoptosis in IEC-6 cells. The modulatory effects of catalpol on apoptosis through inhibiting JAK2/STAT3 pathway observed in vivo were further evaluated by using H/R-injured IEC-6 cells. Both TUNEL assay and AO/EB staining showed that apoptosis was increased in H/ R-injured IEC-6 cells, and catalpol (10, 20, 40 mM) pretreatment reversed the increased apoptosis compared with H/R-injured IEC-6 cells (Fig. 5D and E). And western blot analysis showed that catalpol (10, 20, 40 mM) pretreatment increased Bcl-2/Bax ratio and decreased the expression of pro-apoptotic cleaved caspase3 compared with control H/R-injured cells (Fig. 6B). Catalpol (40 mM) did not affect those in normal IEC-6 cells.

Modulation of catalpol on JAK2/STAT3 signaling pathway
JAK2 and STAT3 phosphorylation in IEC-6 cells in H/R condition was signicantly increased. Catalpol pretreatment signicantly and selectively reversed the increased phosphorylation of JAK2 and STAT3 in a dose-dependent manner (Fig. 6A). Immunouorescence analysis indicated that p-STAT3 nuclear translocation was inhibited by catalpol (40 mM) (Fig. 6C). IEC-6 cells transfected with JAK2 siRNA assayed in H/R condition were used to assess the correlation between catalpol-induced protection and JAK2/STAT3 signaling pathway. In vitro siRNA knockdown of JAK2 signicantly decreased JAK2, p-JAK2, STAT3, p-STAT3, and other apoptosis-related proteins including Bcl-2, Bax, and cleaved caspase3, indicating that JAK2 siRNA partially simulated the effects of catalpol. JAK2 siRNA + catalpol did not further decrease JAK2, p-JAK2, STAT3, p-STAT3, and other apoptosis-related proteins, suggesting that inhibition of JAK2/STAT3 signaling pathway plays a key role in catalpolinduced protective effects against intestinal I/R injury.

Discussion
Intestinal I/R injury is characterized with a high morbidity and mortality rate. 35,36 Its pathophysiological process is typically associated with intestinal and mesenteric vascular dysfunction, which can also occur as a result of surgery, organ transplantation, septic shock, and trauma. 37 I/R may cause intestinal injury and induce systemic circulation of intestinal bacteria and endogenous endotoxins, leading to multiple organ dysfunction syndrome.
Ischemia causes injury and reperfusion may exacerbate the damage. Intestinal I/R injury can be caused by inammatory cytokines, 38 oxygen free radicals, 39 and neutrophil inltration. 40 And excessive oxidative stress, inammation damage, and disruption of intestinal epithelial homeostasis, ultimately induce apoptosis. [41][42][43][44] Apoptosis is found to have a vital impact in tissue injury and apoptosis is a major mode of cell death induced by I/R. 45,46 Therefore, anti-inammation, antioxidation, restoration of barrier function, and inhibition of apoptosis can be selected as the indicators for amelioration of intestinal I/R injury.
Our in vivo study showed that catalpol (25, 50 mg kg À1 ) pretreatment effectively alleviated intestinal I/R injury by reducing the oxidative stress through reversing the increased MDA level and reversing the decreased SOD and GSH levels in catalpol-treated I/R-injured rats ( Fig. 2A). Catalpol pretreatment markedly alleviated the intestinal lesions (Fig. 1B) and signicantly ameliorated the inammation characterized by reversing the increased MPO, IL-6, IL-1b, TNF-a, and I-FABP (Fig. 2B). And catalpol pretreatment markedly reduced the DAO level and increased the expressions of both occludin and ZO-1 proteins (Fig. 3), showing that catalpol restored intestinal barrier function in intestinal I/R-injured rats. All of these in vivo results indicated that catalpol inhibited both inammation and oxidative stress and restored intestinal barrier function.
Mitochondria play an important role in energy production and reactive oxygen homeostasis, 47 triggering apoptotic events. 48 Our in vivo results indicated that catalpol pretreatment reduced TUNEL-positive cells, increased Bcl-2/Bax ratio and decreased the expression of cleaved caspase3 (Fig. 4A and C), showing that catalpol markedly alleviated intestinal I/R injury by inhibiting apoptosis. The in vivo inhibitory effects of catalpol on apoptosis were also observed in vitro. TUNEL assay (Fig. 5C), AO/EB staining (Fig. 5D), and western blot analysis (Fig. 6A and B) indicated that catalpol signicantly prevented the occurrence of apoptosis.
JAK2/STAT3 signaling pathway plays a major role in cytokine signal transduction of various cytokines and growth factors as IL-6 (ref. 49 and 50) and this signal pathway is activated during the tissue injuries and closely related to oxidative stress, inammation, and apoptosis. 12,51-53 Activation of JAK2/STAT3 signaling pathway was observed in intestinal I/R injury, 12 however the detailed mechanism of JAK2/STAT3 in I/R-induced apoptosis needs to be further explored. Our results from both in vivo and in vitro studies showed that catalpol pretreatment (25, 50 mg kg À1 ) signicantly and selectively reversed the increased p-JAK2 and p-STAT3 without affecting total JAK2 and STAT3 ( Fig. 4B and 6A), indicating that catalpol-induced anti-apoptotic effects on intestinal I/R was mediated through selective inhibiting p-JAK2 and p-STAT3. Furthermore, in vitro study showed that catalpol inhibited p-STAT3 nuclear translocation, resulting in a decrease of apoptosis (Fig. 6C).
To verify the relationship between catalpol-induced protection and JAK2/STAT3 signaling pathway, JAK2 siRNA was transfected into IEC-6 cells under H/R condition. JAK2 siRNA decreased the expression of JAK2, p-JAK2, STAT3, p-STAT3, and inhibited mitochondria-mediated apoptosis (Fig. 7). And JAK2 siRNA + catalpol could not further decrease the expression of JAK2, p-JAK2, and other apoptosis-related proteins (Fig. 7), suggesting that JAK2/STAT3 signaling pathway plays a key role in catalpol-induced anti-apoptotic effect. In summary, the present study demonstrated that catalpol pretreatment signicantly attenuated rat intestinal I/R injury by inhibiting oxidative stress and inammation, restoring intestinal barrier function, and decreasing mitochondria-mediated apoptosis through blocking JAK2/STAT3 signaling pathway via selective inhibition of p-JAK2. Catalpol did not signicantly affect the corresponding controls. Furthermore, catalpol is low toxicity, and has anti-bacterial activity which may inhibit bacterial infection during the development of intestinal I/R injury. Our study suggests that p-JAK2 plays a key role in catalpol-induced protection on intestinal I/R injury and p-JAK2 can be chosen as a therapeutic target, providing valuable information for the further translational studies of catalpol in protecting against intestinal I/R injury.

Conflicts of interest
The authors have declared no conict of interest.