Male Sprague-Dawley rats (Shanghai Laboratory Animal Center, China) aged 7–8 weeks and weighing 250–300 g were used in the experiments. The rats were housed under standard laboratory conditions at a temperature of 20–22°C and 12 h light-dark cycle and given access to food and water. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. The study protocol was approved by the local Ethics Committee of the 117th Hospital of PLA. Rapamycin, AG490, and curcumin were purchased from Sigma-Aldrich (St. Louis, MO, USA). A TNF-α ELISA Kit, IL-1β ELISA Kit, and IL-6 ELISA Kit were purchased from Beyotime Biotechnology (Haimen, China). Antibodies against HMGB1, JAK2, p-JAK2, STAT3, p-STAT3, and the internal standard GAPDH (all from Affinity Biosciences, Cincinnati, OH, USA) were used for western blot analysis and immunohistochemical (IHC) analysis. The secondary antibody was a ready-to-use goat-anti rabbit (or goat-anti-mouse, or donkey-anti-goat) HRP-IgG dilution purchased from Beyotime Biotechnology (Haimen, China).

Rats were fasted overnight before the operation with free access to drinking water. The rats were anesthetized with intraperitoneal injection of anesthetic and fixed in supine position. The hair in the median position of the neck was removed and disinfected, and the skin was covered with dressing. The middle part of the neck was incised to expose the right common carotid artery, internal carotid artery, and extracranial branch (pterygopalatine artery), and the external carotid artery and its branches (occipital artery and its superior thyroid artery). The occipital artery, superior thyroid artery, and pterygopalatine artery were clipped and the occipital artery and superior thyroid artery were sliced off. The distal segment of the external carotid artery was ligated, and with a slipknot at the proximal end. A small hemostatic clamp was used to clamp down the common carotid artery and the internal carotid artery. This was followed by a slight incision with micro scissor on the distal end of external carotid artery (proximal end of ligature). A single-head vein indwelling needle sealed up with heparin was inserted along the small cut all the way to the bifurcation of the common carotid artery. The stylet was removed and the micro hemostatic clamp was removed from the internal carotid artery. After completely drilling the blood and gas, the prepared embolus was slowly injected into the internal carotid artery (preparation of embolus: Collect blood samples and centrifuged at 3,000 rpm for 10 min, followed by addition of CaCl₂ and thrombin. The mixture was injected into an anesthesia catheter and the tube was placed in a 37°C water bath for 15 min. Then, the gel was sliced into 0.8–1.5 mm-long segments. Then, 4–6 segments were selected and placed in 1 ml PBS solution). After complete injection of the embolus, the micro hemostatic clamp was removed from the internal carotid artery and the blood flow was restored for 1 min. Then, the venous indwelling needle was withdrawn, the external carotid artery was ligated, and the neck skin was stitched. The mice in the sham group were given the same treatment, except no embolus was injected. Twenty-four hours after surgery, the rat was incised in the chest under anesthesia to expose the thoracic cavity. The right atrial appendage was cut open and perfused with physiological saline from the left ventricle until clear fluid flowed out. Then, the ischemic tissue of the right cerebrum was collected to prepare tissue homogenate or paraffin sections with 4°C physiological saline in the proportion of 1:10 by weight.

The rat brain tissue homogenate was prepared and centrifuged at 12,000 × g and 4°C for 15 min. The supernatant was collected. Standard control and tissue homogenate was added separately into the well of coated plate. The plate was covered with plate sealing film and incubated at 37°C for 30 min. Then, the plate was washed 5 times, followed by addition of 50 µl of ELISA reagents. Repeat the incubation and washing steps, then add coloring agent 50 µl, coloring agent B 50 µl and finally the termination solution 50 µl to stop the reaction. The absorbance was measured in a microplate reader and a histogram was drawn.

Every 20 mg of rat brain tissue homogenate was mixed with 200 µl lysate. The mixture was treated with homogenizer until complete lysis. Then, the sample was centrifuged at 4°C, 12,000 g for 15 min. The supernatant was collected to measure the protein concentration with BCA kits (Bio-Rad, Hercules, CA, USA). The denatured protein sample was mixed into the sample buffer and added to the sample lanes. After 70 min electrophoresis, the protein was transferred to PVDF membrane (Millipore, Bedford, MA, USA) and blocked with 1X TBST solution containing 5% skim milk for 1 h. This was followed by overnight incubation at 4°C with primary antibody, the membranes were washed 4 times with 1X TBST for 10 min each time. Then, the membrane was incubated with secondary antibody at room temperature for 1 h, then washed 4 times with 1X TBST for 10 min each. Finally the fluorescence was developed, enhanced and fixed with enhanced chemiluminescence ECL kit (Pierce, Rockford, IL, USA).

Paraffin sections were dewaxed and hydrated and placed in EDTA-containing antigen repair buffer (pH 9.0). Antigen retrieval was performed in a microwave oven. The slices were placed in 3% hydrogen peroxide solution to block endogenous peroxidase and blocked with 5% BSA solution at room temperature for 30 min. Then, the slices were incubated with primary antibody at 4°C overnight, followed by incubation with secondary antibody for 50 min at room temperature. Then, the slices were washed with PBS for 15 min. Finally, DAB color developing solution was added dropwise. The development time was controlled by observing the slides under a microscope. Positive staining was indicated with brown spots. The development was terminated by washing with tap water. The nuclei were stained with Harris hematoxylin, and each slice was dehydrated and sealed.

Data are expressed as the mean ± standard error of the mean (SEM) of at least three independent experiments. Standard error bars were included for all data points. Statistical analysis was performed using Student's t-test when only two groups were present or a one-way analysis of variance followed by the Student-Newman-Keuls test when more than two groups were compared. Statistical analyses were conducted with SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered significant.

Rats were allocated into two groups, a sham group and a MCAO group, with 5 rats in each group. ELISA was performed to detect the TNF-α change in cerebral tissue in each group. As indicated in Fig. 1. The amount of TNF-α in the brain tissue homogenate of MCAO group was significantly higher than in the sham group (P<0.01).

Rats were allocated into two groups, a sham group and MCAO group, with 3 rats in each group. The expression of HMGB1, JAK2, p-JAK2, STAT3, and p-STAT3 were assessed by western blot analysis in the brain tissue of the rats in both groups. As indicated in Fig. 2, the concentration of HMGB1 protein in MCAO group was significantly higher than in the sham group (P<0.01). The relative levels of p-JAK2/JAK2 and p-STAT3/STAT3 in brain tissue homogenate of MCAO group were significantly higher than in the sham group (P<0.01), suggesting that cerebral ischemia activates the JAK2/STAT3 signaling pathway and promotes HMGB1 overexpression. There remains some question regarding whether the JAK2/STAT3 signaling pathway is involved in HMGB1 induction and its induced inflammatory response.

Rats were allocated into 5 groups of 3 rats each: a cerebral ischemia group (MCAO), inhibitor-treated group (MCAO + saline), STAT3 inhibitor-treated group (MCAO + RPM), JAK2 inhibitor-treated group (MCAO + AG490), and curcumin-treated group (MCAO + curcumin). The expression of TNF-α, IL-1β, and IL-6 in brain tissue of four groups were detected by ELISA. As indicated in Fig. 3, the concentrations of TNF-α, IL-1β, and IL-6 in cerebral tissue homogenate of MCAO group and MCAO + saline group were comparable (P>0.05). Compared with MCAO + saline group, the contents of TNF-α, IL-1β, IL-6 in MCAO+RPM group, MCAO + AG490 group, and MCAO + curcumin group were significantly lower than in the MCAO + saline group (P<0.01), suggesting that inhibition of JAK2/STAT3 signaling pathway such as curcumin reduces the release of inflammatory factors after cerebral ischemia.

Effects of rapamycin, AG490, and curcumin on the expression of HMGB1, JAK2, p-JAK2, STAT3, and p-STAT3 in brain tissue of rats with cerebral ischemia. The expression levels of HMGB1, JAK2, p-JAK2, STAT3, and p-STAT3 were detected by Western Blot and immunohistochemistry. As indicated in Fig. 4, the expression of HMGB1, JAK2, p-JAK2, STAT3, and p-STAT3 in the brain tissue homogenate of the MCAO + saline group was comparable to that of the MCAO group (P>0.05). The concentrations of p-JAK2/JAK2 and p-STAT3/STAT3 in MCAO + RPM group, MCAO + AG490 group, and MCAO + curcumin group were significantly lower than in the MCAO + saline group (P<0.01), suggesting that inhibition of JAK2/STAT3 signaling pathway such as curcumin can reduce the expression of HMGB1 in ischemic brain tissue.