Male SD rats were randomly divided into a sham group, a model group, high-, medium-, and low-dose chrysin groups (200, 100, and 50 mg/kg), and a positive drug group (Ginaton, 21.6 mg/kg). The CIRI model was induced in rats by transient middle cerebral artery occlusion (tMCAO). The indexes were evaluated and the samples were taken 24 h after the operation.

Rats were anesthetized by pentobarbital sodium at a dosage of 40 mg/kg by intraperitoneal injection. Rats were first anchored on to an operating table in the supine position. The fur around the incision was shaved and then disinfected. Subsequently, the neck of each rat was incised in the middle to expose the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). The proximal end of the CCA and ECA were ligated and severed using a 0.285 mm nylon suture. The suture was inserted from the ECA stump through the ICA to reach the MCA. The MCA was then occluded for 2 h to create ischemic conditions. Next, the nylon suture was slowly pulled out to restore blood flow and simulate reperfusion condition.

Rats were anesthetized by pentobarbital sodium at a dosage of 40 mg/kg by intraperitoneal injection. Rats were first anchored on to an operating table in the supine position. The fur around the incision was shaved and then disinfected. Subsequently, the neck of each rat was incised in the middle to expose the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). The proximal end of the CCA and ECA were ligated and severed using a 0.285 mm nylon suture. The suture was inserted from the ECA stump through the ICA to reach the MCA. The MCA was then occluded for 2 h to create ischemic conditions. Next, the nylon suture was slowly pulled out to restore blood flow and simulate reperfusion condition.

A total of 108 gerbils (male; body weight, 70-90 g; age, 12 to 16 weeks) were used in this study. The gerbils were randomly divided into the following five groups: the vehicle-treated group (sham group),which was given an equal volume of physiological saline; the carvacrol (CAR)-treated group (CAR group); the model group, which underwent the ligation of the bilateral carotid artery for 5 min followed by the loosening of the arterial clamp for reperfusion; the model + CAR-treated groups, which included the CAR-treated group and the model + CAR-treated groups that were treated with CAR (25, 50 and 100 mg/kg/day, i.p.) for 2 consecutive weeks and the model + DFO-treated groups that were treated with DFO (150 mg/kg/day, i.p.) for 2 consecutive weeks as the positive drug group.

Male SD rats were randomly divided into a sham group, a model group, high-, medium-, and low-dose chrysin groups (200, 100, and 50 mg/kg), and a positive drug group (Ginaton, 21.6 mg/kg). The CIRI model was induced in rats by transient middle cerebral artery occlusion (tMCAO). The indexes were evaluated and the samples were taken 24 h after the operation.

The mice (23-25 g, 8-10 weeks old) were subjected to transientmiddle cerebral artery occlusion (tMCAO) to induce cerebral ischemia as previously described protocol . Briefly, mice were anesthetized with intraperitoneal injection of pentobarbital sodium (60 mg/kg) and subcutaneous injection of meloxicam (10mg/kg) during tMCAO operation. Monofilament with a silicon coating on the tip and a diameter of 0.12 mm (A5-122, Beijing Cinontech Co. Ltd., China) was inserted into the ICA from CCA to occlude the middle cerebral artery (MCA) for 1.5 h. The suture was then removed to restore blood flow for another 22.5 h reperfusion. Sham control mice were subjected to similar surgical operations without MCA occlusion. Specifically, the monofilament was inserted only 5 mm above the carotid bifurcation and withdrew immediately in the Sham group.

Specific pathogen-free adult male SD rats, (80 ± 5) days old and weighing 220-250 g, were provided by the Hunan Slack Jingda Experimental Animal Co., Ltd (Hunan, China). SD rats were randomly divided into 4 groups with 15 in each group: sham operation group, MCAO group, MCAO + DFP group and MCAO + NTE group. The rats were treated with drugs via oral gavage. According to the average body weight, the MCAO + NTE rats were given NTE at 27 g/kg, and the sham operation and the MCAO rats were given the same volume of saline (2.5 mL) for 7 consecutive days. The MCAO + DFP rats were given DFP at a dose of 125 mg/kg for 3 consecutive days.

Male C57BL/6 mice weighing 20-25 g each were obtained from the Animal Experimental Center of Yisi (Changchun, China). Mice were group-housed in a 12 h light/dark cycle (light between 08:00 and 20:00 h) in a temperature-controlled environment room (23-25 ). Mice had ad libitum access to food and water. All surgical procedures were carried out on animals anesthetized with sodium pentobarbital (30 mg/kg) via intraperitoneal injection. MCAO was achieved by inserting a silicone rubber-coated nylon monofilament into the internal carotid artery through the external carotid artery and temporary ligation of the right common carotid artery with a suture. After 45 min of ischemia, blood flow was restored by removing the filament and the suture, and the mice were allowed to recover for 24 h.

Rats were randomly divided into the Sham, middle cerebral artery occlusion-reperfusion (MCAO/R), and MCAO/R + AST IV (28 mg/kg) groups. The MCAO/R + AST IV group was intragastrically injected with 10 mL/kg AST IV at 50, 26, and 2 h before modelling (Xiao et al., 2021). The Sham and MCAO/R groups received equal amounts of normal saline. As described previously, the modified Longa method (Longa et al., 1989) was used to establish the MCAO/R model. After anaesthesia with 2%sodium pentobarbital, the left common carotid artery(CCA), the external carotid artery(ECA), and the internal carotid artery(ICA) were isolated. The distal end of the ECA was ligated, a small incision was made at the stump of the ECA, and a suture (Batch number: 2636A2, Beijing Seinong Technology Co., Ltd., Beijing, China; head-end diameter: 0.36 ± 0.02 mm) was inserted into the ICA from the ECA through the bifurcation of the CCA. To achieve cerebral ischaemia, the head-end was used to block blood flow in the middle cerebral artery until the intracranial segment of the ICA was inserted. The suture was removed after 2 h, and follow-up experiments were performed 24 h after reperfusion. In the Sham group, the CCA, ECA, and ICA were exposed and separated, but no sutures were inserted. Penicillin powder was used to fight infection after operation.

Rats were randomly divided into the Sham, middle cerebral artery occlusion-reperfusion (MCAO/R), and MCAO/R + AST IV (28 mg/kg) groups. The MCAO/R + AST IV group was intragastrically injected with 10 mL/kg AST IV at 50, 26, and 2 h before modelling (Xiao et al., 2021). The Sham and MCAO/R groups received equal amounts of normal saline. As described previously, the modified Longa method (Longa et al., 1989) was used to establish the MCAO/R model. After anaesthesia with 2%sodium pentobarbital, the left common carotid artery(CCA), the external carotid artery(ECA), and the internal carotid artery(ICA) were isolated. The distal end of the ECA was ligated, a small incision was made at the stump of the ECA, and a suture (Batch number: 2636A2, Beijing Seinong Technology Co., Ltd., Beijing, China; head-end diameter: 0.36 ± 0.02 mm) was inserted into the ICA from the ECA through the bifurcation of the CCA. To achieve cerebral ischaemia, the head-end was used to block blood flow in the middle cerebral artery until the intracranial segment of the ICA was inserted. The suture was removed after 2 h, and follow-up experiments were performed 24 h after reperfusion. In the Sham group, the CCA, ECA, and ICA were exposed and separated, but no sutures were inserted. Penicillin powder was used to fight infection after operation.

Seventy-three specific-pathogen-free (SPF)grade healthy male Sprague Dawley (SD) rats, weighing 240 ± 20 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., China (animal certificate number SCXK (Xiang) 2013-0004). The animals were reared in an SPF animal laboratory, and the ambient temperature was maintained at 23 ± 1 . All protocols followed the ARRIVE guidelines in terms of study design, sample size, randomization, outcome measures, data analysis, experimental procedures, and reporting of results. This study was approved by the Animal Ethics Committee of the Hunan University of Chinese Medicine.

Rats were randomly assigned to six groups: (1) the sham group, (2) the middle cerebral artery ischaemia-occlusion-reperfusion (MCAO/R) group, (3) the AST IV group, (4) the PNS group, (5) the combination group and (6) the combination + brusatol group. One hundred rats were used in the experiment, of which 9 died during surgery, 10 died of intracranial haemorrhage and brain injury and 63 rats were successfully modelled, for a final success rate of 76.8%. Each group included 9 rats. Behavioural testing was performed on 5 animals in each group. After behavioural testing, 3 rats were used for TTC staining and 6 were used for kit detection and western blot analysis. Existing studies have revealed the toxicological effects of the compatibility of astragalus and P. notoginseng. The dosage and method of AST IV (28 mg/kg) and PNS (80 mg/kg) alone or in combination have been previously determined and were administered intragastrically for three consecutive days (10 ml/kg each time), and the optimal administration times were 50, 26 and 2 h before model establishment.Brusatol (1 mg/kg) was administered intraperitoneally for 1 h prior to modelling. The sham group and the MCAO/R group were given the same amount of saline.

Rats were anesthetized withisoflurane(2-3% oxygen) and placed in asupine position. And theright common carotid artery (CCA),external carotid artery (ECA), andinternal carotid artery (ICA) were exposed in sequence and separated carefully. Then we ligated the CCA and the ECA in turn, and at the same time, we clamped the internal carotid artery with an arterial clamp. Finally, we inserted a silicone nylon monofilament from the CCA into themiddle cerebral arteryand temporarily fixed it. After 1.5 h ofischemia, the monofilament was taken out and the blood vessels were ligated at theincision. The neck wound was sutured with surgical sutures. Subsequent experiments were performed after 12 h ofreperfusion. In thesham operationrats, except for the absence of the monofilament, the sham operation rats underwent the same surgical procedures as the MCAO/R model rats.

Rats were randomly assigned to six groups: (1) the sham group, (2) the middle cerebral artery ischaemia-occlusion-reperfusion (MCAO/R) group, (3) the AST IV group, (4) the PNS group, (5) the combination group and (6) the combination + brusatol group. One hundred rats were used in the experiment, of which 9 died during surgery, 10 died of intracranial haemorrhage and brain injury and 63 rats were successfully modelled, for a final success rate of 76.8%. Each group included 9 rats. Behavioural testing was performed on 5 animals in each group. After behavioural testing, 3 rats were used for TTC staining and 6 were used for kit detection and western blot analysis. Existing studies have revealed the toxicological effects of the compatibility of astragalus and P. notoginseng. The dosage and method of AST IV (28 mg/kg) and PNS (80 mg/kg) alone or in combination have been previously determined and were administered intragastrically for three consecutive days (10 ml/kg each time), and the optimal administration times were 50, 26 and 2 h before model establishment.Brusatol (1 mg/kg) was administered intraperitoneally for 1 h prior to modelling. The sham group and the MCAO/R group were given the same amount of saline.

Male C57BL/6 mice weighing 20-25 g each were obtained from the Animal Experimental Center of Yisi (Changchun, China). Mice were group-housed in a 12 h light/dark cycle (light between 08:00 and 20:00 h) in a temperature-controlled environment room (23-25 ). Mice had ad libitum access to food and water. All surgical procedures were carried out on animals anesthetized with sodium pentobarbital (30 mg/kg) via intraperitoneal injection. MCAO was achieved by inserting a silicone rubber-coated nylon monofilament into the internal carotid artery through the external carotid artery and temporary ligation of the right common carotid artery with a suture. After 45 min of ischemia, blood flow was restored by removing the filament and the suture, and the mice were allowed to recover for 24 h.

Adult male SD rats (250-300 g) were purchased from Charies River (Beijing, China). The animals were placed in laboratory cages, kept on a 12-h light-dark cycle, and had free access to food and water throughout the study. The rats were randomly assigned to the sham (only the left neck was exposed without ligation) group, MACO group, and sevo + MACO (2.5% sevoflurane before refusion) group. The MCAO model was made by a modified nylon suture method. After 1 h of ischemia, the suture was gently pulled to the beginning of the external carotid artery and re-perfused for 24 h. For sevoflurane postconditioning, rats were stabilized in a gas-tight anesthesia chamber with sevoflurane inhalation for 1 h at the onset of blood refusion. Sevoflurane (AbbVie, Japan) was delivered at a concentration of 2.5% through a vaporizer (Vapor 2000, Germany). In the sham or MCAO group, rats were only exposed to the mixed gas (95% O2 and 5% CO2).

Male SD rats were randomly divided into a sham group, a model group, high-, medium-, and low-dose chrysin groups (200, 100, and 50 mg/kg), and a positive drug group (Ginaton, 21.6 mg/kg). The CIRI model was induced in rats by transient middle cerebral artery occlusion (tMCAO). The indexes were evaluated and the samples were taken 24 h after the operation.

The mice (23-25 g, 8-10 weeks old) were subjected to transientmiddle cerebral artery occlusion (tMCAO) to induce cerebral ischemia as previously described protocol . Briefly, mice were anesthetized with intraperitoneal injection of pentobarbital sodium (60 mg/kg) and subcutaneous injection of meloxicam (10mg/kg) during tMCAO operation. Monofilament with a silicon coating on the tip and a diameter of 0.12 mm (A5-122, Beijing Cinontech Co. Ltd., China) was inserted into the ICA from CCA to occlude the middle cerebral artery (MCA) for 1.5 h. The suture was then removed to restore blood flow for another 22.5 h reperfusion. Sham control mice were subjected to similar surgical operations without MCA occlusion. Specifically, the monofilament was inserted only 5 mm above the carotid bifurcation and withdrew immediately in the Sham group.

Male SD rats were randomly divided into a sham group, a model group, high-, medium-, and low-dose chrysin groups (200, 100, and 50 mg/kg), and a positive drug group (Ginaton, 21.6 mg/kg). The CIRI model was induced in rats by transient middle cerebral artery occlusion (tMCAO). The indexes were evaluated and the samples were taken 24 h after the operation.

Seventy-three specific-pathogen-free (SPF)grade healthy male Sprague Dawley (SD) rats, weighing 240 ± 20 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., China (animal certificate number SCXK (Xiang) 2013-0004). The animals were reared in an SPF animal laboratory, and the ambient temperature was maintained at 23 ± 1 . All protocols followed the ARRIVE guidelines in terms of study design, sample size, randomization, outcome measures, data analysis, experimental procedures, and reporting of results. This study was approved by the Animal Ethics Committee of the Hunan University of Chinese Medicine.

Wild-type SD rats were kept in the Animal Experiment Center of Southeast University. Experimental rats were divided into 4 groups (n = 6 per group). The method of establishing the I/R model was provided in supplementary material. Then, we covered the ligation with gel. In order to fully cover the infarcted area of the heart, we chose to inject about 300 uL of mimics + Gel at 23 mm below the left atrial appendage (about the ligation). In order to prevent excessive irradiation of tissue burns, we selected each irradiation for 2 min to control the body surface temperature for a total of 10 min of irradiation.

Specific pathogen-free adult male SD rats, (80 ± 5) days old and weighing 220-250 g, were provided by the Hunan Slack Jingda Experimental Animal Co., Ltd (Hunan, China). SD rats were randomly divided into 4 groups with 15 in each group: sham operation group, MCAO group, MCAO + DFP group and MCAO + NTE group. The rats were treated with drugs via oral gavage. According to the average body weight, the MCAO + NTE rats were given NTE at 27 g/kg, and the sham operation and the MCAO rats were given the same volume of saline (2.5 mL) for 7 consecutive days. The MCAO + DFP rats were given DFP at a dose of 125 mg/kg for 3 consecutive days.

Wild-type SD rats were kept in the Animal Experiment Center of Southeast University. Experimental rats were divided into 4 groups (n = 6 per group). The method of establishing the I/R model was provided in supplementary material. Then, we covered the ligation with gel. In order to fully cover the infarcted area of the heart, we chose to inject about 300 uL of mimics + Gel at 23 mm below the left atrial appendage (about the ligation). In order to prevent excessive irradiation of tissue burns, we selected each irradiation for 2 min to control the body surface temperature for a total of 10 min of irradiation.

Rats were anesthetized withisoflurane(2-3% oxygen) and placed in asupine position. And theright common carotid artery (CCA),external carotid artery (ECA), andinternal carotid artery (ICA) were exposed in sequence and separated carefully. Then we ligated the CCA and the ECA in turn, and at the same time, we clamped the internal carotid artery with an arterial clamp. Finally, we inserted a silicone nylon monofilament from the CCA into themiddle cerebral arteryand temporarily fixed it. After 1.5 h ofischemia, the monofilament was taken out and the blood vessels were ligated at theincision. The neck wound was sutured with surgical sutures. Subsequent experiments were performed after 12 h ofreperfusion. In thesham operationrats, except for the absence of the monofilament, the sham operation rats underwent the same surgical procedures as the MCAO/R model rats.

All animal experiments described in this study were carried out in accordance with the U.K. Animals (Scientific Procedures) Act and were approved by the Experimental Animal Center of Wenzhou Medical University (No. wydw2022-0032). Six-to-eight-weeks old male ICR mice were obtained from Beijing Weitonglihua Experimental Animal Technology Co. Ltd. (Beijing, China). Mice were group-housed in the breeding environment under a 12/12h light/dark cycle, controlled temperature of 20-22 and 50-60% humidity with ad libitum chow and water. All animals were randomized for the research and procedures.

The mice (23-25 g, 8-10 weeks old) were subjected to transientmiddle cerebral artery occlusion (tMCAO) to induce cerebral ischemia as previously described protocol . Briefly, mice were anesthetized with intraperitoneal injection of pentobarbital sodium (60 mg/kg) and subcutaneous injection of meloxicam (10mg/kg) during tMCAO operation. Monofilament with a silicon coating on the tip and a diameter of 0.12 mm (A5-122, Beijing Cinontech Co. Ltd., China) was inserted into the ICA from CCA to occlude the middle cerebral artery (MCA) for 1.5 h. The suture was then removed to restore blood flow for another 22.5 h reperfusion. Sham control mice were subjected to similar surgical operations without MCA occlusion. Specifically, the monofilament was inserted only 5 mm above the carotid bifurcation and withdrew immediately in the Sham group.

Male SD rats were randomly divided into a sham group, a model group, high-, medium-, and low-dose chrysin groups (200, 100, and 50 mg/kg), and a positive drug group (Ginaton, 21.6 mg/kg). The CIRI model was induced in rats by transient middle cerebral artery occlusion (tMCAO). The indexes were evaluated and the samples were taken 24 h after the operation.

Male gerbils weighing 70-90 g (12 weeks) were selected for this study. Gerbils were anesthetized with 7% chloral hydrate (350 mg/kg) and the bilateral common carotid arteries were occluded using artery clips. After 5 min, the clips were removed to restore cerebral blood flow. After the operation, place the gerbil on an electric blanket to keep the gerbil's body temperature. The sham group underwent the same surgical procedure without ligation of carotid arteries and was given an equal volume of physiological saline as in the treated groups. The model + galangin (Jiangsu Yongjian Pharmaceutical Technology, 548-83-4, Purity: >=98% (HPLC)) groups underwent the same procedure as the model group and then were received galangin at 25, 50, or 100 mg/kg/day for two continuous weeks.

The male Sprague Dawley rats were randomly divided into control group, sham group, middle cerebral artery occlusion/reperfusion model group (MCAO/R group) (according to different ischemia reperfusion time, the model group was divided into the 3, 6, 12 and 24 hours reperfusion groups after 2 hour-ischemia), and vehicle group, agomir-27a group, antagomir-27a group. The Zea-Longa method was used to establish rat MCAO model.

Wild-type SD rats were kept in the Animal Experiment Center of Southeast University. Experimental rats were divided into 4 groups (n = 6 per group). The method of establishing the I/R model was provided in supplementary material. Then, we covered the ligation with gel. In order to fully cover the infarcted area of the heart, we chose to inject about 300 uL of mimics + Gel at 23 mm below the left atrial appendage (about the ligation). In order to prevent excessive irradiation of tissue burns, we selected each irradiation for 2 min to control the body surface temperature for a total of 10 min of irradiation.

Wild-type SD rats were kept in the Animal Experiment Center of Southeast University. Experimental rats were divided into 4 groups (n = 6 per group). The method of establishing the I/R model was provided in supplementary material. Then, we covered the ligation with gel. In order to fully cover the infarcted area of the heart, we chose to inject about 300 uL of mimics + Gel at 23 mm below the left atrial appendage (about the ligation). In order to prevent excessive irradiation of tissue burns, we selected each irradiation for 2 min to control the body surface temperature for a total of 10 min of irradiation.