All animals were purchased from the Animal Experimental Center of Wuhan University (ABLS-III Laboratory). C57BL/6 male mice weighing 20-25 g were used for this study. HK-2 cells were seeded into 96-well plates (5 x 10⁵ cells/well) and cultured for 24 h until 80% confluence. Subsequently, we have added LPS (10 ug/ml) into the cultured cells for 22 h to establish the cell model of LPS-induced AKI.

Briefly, female BALB/c mice (6-8 weeks, weighing 20-25 g, purchased from Hunan SJA Laboratory Animal Co., Ltd., Changsha, Hunan, China) were raised in specific pathogen-free conditions under controlled temperature (23-25 ) and humidity (40-80%) as well as a 12 h dark/light cycle for 1 week of acclimation. They were fed a standard chow diet and waterad libitum. Mice were anaesthetised with 2% isoflurane inhalation and underwent moderate caecal ligation and puncture in accordance with a previously reported protocol (Rittirsch etal.2009). Meanwhile, mice in the control groups were subjected to a sham operation. Buprenorphin (0.05 mg/kg) was injected for postoperative analgesia, and all mice were placed in cages immediately after the surgical procedures with free access to water and food (Rittirsch etal. 2009).

Eight-week-old wild-type (WT) and Nrf2-knockout (Nrf2-/-) littermate male mice on a C57BL/6J background were purchased from Cyagen (Suzhou, China.) and maintained at the Centre for Animals of Wuhan University (Wuhan, China). Before the experiment, the mice were separated and given light and dark cycles for 12 h, 22 ± 0.5 temperature, 60 ± 10% humidity, and free accessed to food and water for at least 1 week. Mice were randomly distributed into sham, CLP, CLP + Irisin (Ir group) and CLP + Irisin + Era (Ir + Era group) groups.

Male C57BL/6 mice aged 8-12 weeks, weighing about 25 g, were purchased from Shanghai Experimental Animal Center of China. The mice were anesthetized with isoflurane. The abdomen area was disinfected after shaving. A 1 cm midline incision was made on the lower abdomen skin to expose the cecum. A 5-0 silk thread was used to ligate the midpoint between the distal pole and the bottom of the cecum. A 21 g needle was used to penetrate the cecum twice from the mesenteric toward the antimesenteric direction. After the wound was sutured, 0.25% ropivacaine was injected subcutaneously for postoperative analgesia. The mice were immediately resuscitated by injecting prewarmed normal saline 1 ml subcutaneously.

Clean male C57BL/6 mice (8-10 wk old, weighing 25-29 g) were purchased from Beijing HFK Bioscience Co. Ltd. The 90 mice were randomly assigned into nine groups after 1 wk of adaptive feeding with 10 mice in each group: normal group (intraperitoneally injected with the same amount of PBS), LPS group [intraperitoneally injected with 15 mg/kg LPS (Sigma-Aldrich, St. Louis, Cat. No. L2880)], LPS + Rsv group (intraperitoneally injected with 15 mg/kg LPS and pretreated with 50 mg/kg resveratrol; 30), LPS + antagomiR-NC group [intraperitoneally injected with 15 mg/kg LPS, and simultaneously injected with 0.4 pmol/uL antagomiR-NC group (Merck, Darmstadt, Germany)], LPS + miR-149 antagomiR group (intraperitoneally injected with 15 mg/kg LPS, and simultaneously injected with 0.4 pmol/uL miR-149 antagomiR), LPS + Rsv + miR-149 antagomiR group (intraperitoneally injected with 15 mg/kg LPS, pretreated with 50 mg/kg resveratrol, and simultaneously injected with 0.4 pmol/uL miR-149 antagomiR), LPS + Fer-1 group (intraperitoneally injected with 15 mg/kg LPS, and simultaneously injected with 2.5 umol/kg ferroptosis inhibitor ferrostatin-1), and LPS + Rsv + Fer-1 group (intraperitoneally injected with 15 mg/kg LPS, pretreated with 50 mg/kg resveratrol, and simultaneously injected with 2.5 umol/kg ferroptosis inhibitor ferrostatin-1.

Clean male C57BL/6 mice (8-10 wk old, weighing 25-29 g) were purchased from Beijing HFK Bioscience Co. Ltd. The 90 mice were randomly assigned into nine groups after 1 wk of adaptive feeding with 10 mice in each group: normal group (intraperitoneally injected with the same amount of PBS), LPS group [intraperitoneally injected with 15 mg/kg LPS (Sigma-Aldrich, St. Louis, Cat. No. L2880)], LPS + Rsv group (intraperitoneally injected with 15 mg/kg LPS and pretreated with 50 mg/kg resveratrol; 30), LPS + antagomiR-NC group [intraperitoneally injected with 15 mg/kg LPS, and simultaneously injected with 0.4 pmol/uL antagomiR-NC group (Merck, Darmstadt, Germany)], LPS + miR-149 antagomiR group (intraperitoneally injected with 15 mg/kg LPS, and simultaneously injected with 0.4 pmol/uL miR-149 antagomiR), LPS + Rsv + miR-149 antagomiR group (intraperitoneally injected with 15 mg/kg LPS, pretreated with 50 mg/kg resveratrol, and simultaneously injected with 0.4 pmol/uL miR-149 antagomiR), LPS + Fer-1 group (intraperitoneally injected with 15 mg/kg LPS, and simultaneously injected with 2.5 umol/kg ferroptosis inhibitor ferrostatin-1), and LPS + Rsv + Fer-1 group (intraperitoneally injected with 15 mg/kg LPS, pretreated with 50 mg/kg resveratrol, and simultaneously injected with 2.5 umol/kg ferroptosis inhibitor ferrostatin-1.

Beijing Vital River Laboratory Animal Technology Co. Ltd. provided 20 specific pathogen-free female C57BL/6J mice (8-10 weeks old, weighing 20-22g). All procedures on mice were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. 12 h after surgery, the blood was collected from orbital sinus.

Beijing Vital River Laboratory Animal Technology Co. Ltd. provided 20 specific pathogen-free female C57BL/6J mice (8-10 weeks old, weighing 20-22g). All procedures on mice were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. 12 h after surgery, the blood was collected from orbital sinus.

Beijing Vital River Laboratory Animal Technology Co. Ltd. provided 20 specific pathogen-free female C57BL/6J mice (8-10 weeks old, weighing 20-22g). All procedures on mice were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. 12 h after surgery, the blood was collected from orbital sinus.

Thirty specific-pathogen-free (SPF) male C57BL/6 rats at 8 weeks weighing 200-250 g were obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). The rats were housed under room temperatures (25 ± 2 and 12-h light/dark cycle) and given water and food ad libitum before the trial. Then, they were randomly assigned into the control group (n = 10), model group (n = 10), and model + miR-9-5p angomir group (n = 10). All animal procedures were performed under the Care and Use of Laboratory Animals guidelines and approved by the Guangdong Academy of Medical Sciences. According to a previous study, sepsis was induced by the cecal ligation and puncture (CLP) method. Briefly, we anesthetized the rats with 5% chloral hydrate (0.6 ml/100 g body weight) and made a 1.5 cm midline incision on the anterior abdomen to expose the cecum. Then, the cecum was ligated at 30%, punctured twice with a No.4 surgical needle to extrude the fecal content. Finally, 1 ml of normal saline was used for resuscitation. The rats in miR-9-5p angomir group were injected with 100 ng/ul miR-9-5p angomir (RiboBio, Guangzhou, China) into caudal vein (model + miR-9-5p angomir). The rats in the control group experienced the same procedure without ligation and puncture.

Thirty specific-pathogen-free (SPF) male C57BL/6 rats at 8 weeks weighing 200-250 g were obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Briefly, we anesthetized the rats with 5% chloral hydrate (0.6 ml/100 g body weight) and made a 1.5 cm midline incision on the anterior abdomen to expose the cecum. Then, the cecum was ligated at 30%, punctured twice with a No.4 surgical needle to extrude the fecal content. Finally, 1 ml of normal saline was used for resuscitation. The rats in miR-9-5p angomir group were injected with 100 ng/ul miR-9-5p angomir (RiboBio, Guangzhou, China) into caudal vein (model + miR-9-5p angomir). The rats in the control group experienced the same procedure without ligation and puncture.

Thirty specific-pathogen-free (SPF) male C57BL/6 rats at 8 weeks weighing 200-250 g were obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). The rats were housed under room temperatures (25 ± 2 and 12-h light/dark cycle) and given water and food ad libitum before the trial. Then, they were randomly assigned into the control group (n = 10), model group (n = 10), and model + miR-9-5p angomir group (n = 10). All animal procedures were performed under the Care and Use of Laboratory Animals guidelines and approved by the Guangdong Academy of Medical Sciences. According to a previous study, sepsis was induced by the cecal ligation and puncture (CLP) method. Briefly, we anesthetized the rats with 5% chloral hydrate (0.6 ml/100 g body weight) and made a 1.5 cm midline incision on the anterior abdomen to expose the cecum. Then, the cecum was ligated at 30%, punctured twice with a No.4 surgical needle to extrude the fecal content. Finally, 1 ml of normal saline was used for resuscitation. The rats in miR-9-5p angomir group were injected with 100 ng/ul miR-9-5p angomir (RiboBio, Guangzhou, China) into caudal vein (model + miR-9-5p angomir). The rats in the control group experienced the same procedure without ligation and puncture.

Thirty specific-pathogen-free (SPF) male C57BL/6 rats at 8 weeks weighing 200-250 g were obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Briefly, we anesthetized the rats with 5% chloral hydrate (0.6 ml/100 g body weight) and made a 1.5 cm midline incision on the anterior abdomen to expose the cecum. Then, the cecum was ligated at 30%, punctured twice with a No.4 surgical needle to extrude the fecal content. Finally, 1 ml of normal saline was used for resuscitation. The rats in miR-9-5p angomir group were injected with 100 ng/ul miR-9-5p angomir (RiboBio, Guangzhou, China) into caudal vein (model + miR-9-5p angomir). The rats in the control group experienced the same procedure without ligation and puncture.

A total of 32 male C57BL/6 mice (25 g, 8 weeks old) were obtained from the Guangdong Medical Lab Animal Center and housed in the Laboratory Animal Service Center (Jinan University, Guangdong, China). Mice were anesthetized with isoflurane (RWD Life Science) inhalation at the concentration of 2.5% for anesthetic induction and then at 1% for anesthetic maintenance until the end of the CLP. During the experiment, the body temperature was kept at 36-38 with a heating pad. Anesthetized mice were subjected to midline laparotomy. The cecum was carefully separated to avoid blood vessels damage and the cecum was identified and punctured twice with a 22-gauge needle. Then, the abdominal cavity was closed with two epithelium layers, followed by a normal saline injection subcutaneously for resuscitation before mice were returned to the cage.

Healthy male C57BL/6J mice, weighing 22-24 g, 6 weeks old, were purchased from Tianyao Biotechnology Company (Tianjin, China) and housed in an environment free of specific pathogenic bacteria: temperature 22-24 , relative humidity 50%-70%, alternating day and night every 12 h, and free access to water. The cecal ligation and puncture (CLP) approach was used to establish septic mouse models. The survival rates for 7 days were determined. The Morris water maze (MWM) was utilized to assess cognitive function. Hematoxylin and eosin (HE) staining identified histopathologic alterations in hippocampal tissue.

Eight-week-old wild-type (WT) and Nrf2-knockout (Nrf2-/-) littermate male mice on a C57BL/6J background were purchased from Cyagen (Suzhou, China.) and maintained at the Centre for Animals of Wuhan University (Wuhan, China). Before the experiment, the mice were separated and given light and dark cycles for 12 h, 22 ± 0.5 temperature, 60 ± 10% humidity, and free accessed to food and water for at least 1 week. Mice were randomly distributed into sham, CLP, CLP + Irisin (Ir group) and CLP + Irisin + Era (Ir + Era group) groups.

A total of 32 male C57BL/6 mice (25 g, 8 weeks old) were obtained from the Guangdong Medical Lab Animal Center and housed in the Laboratory Animal Service Center (Jinan University, Guangdong, China). Mice were anesthetized with isoflurane (RWD Life Science) inhalation at the concentration of 2.5% for anesthetic induction and then at 1% for anesthetic maintenance until the end of the CLP. During the experiment, the body temperature was kept at 36-38 with a heating pad. Anesthetized mice were subjected to midline laparotomy. The cecum was carefully separated to avoid blood vessels damage and the cecum was identified and punctured twice with a 22-gauge needle. Then, the abdominal cavity was closed with two epithelium layers, followed by a normal saline injection subcutaneously for resuscitation before mice were returned to the cage.

Briefly, female BALB/c mice (6-8 weeks, weighing 20-25 g, purchased from Hunan SJA Laboratory Animal Co., Ltd., Changsha, Hunan, China) were raised in specific pathogen-free conditions under controlled temperature (23-25 ) and humidity (40-80%) as well as a 12 h dark/light cycle for 1 week of acclimation. They were fed a standard chow diet and waterad libitum. Mice were anaesthetised with 2% isoflurane inhalation and underwent moderate caecal ligation and puncture in accordance with a previously reported protocol (Rittirsch etal.2009). Meanwhile, mice in the control groups were subjected to a sham operation. Buprenorphin (0.05 mg/kg) was injected for postoperative analgesia, and all mice were placed in cages immediately after the surgical procedures with free access to water and food (Rittirsch etal. 2009).

C57BL/6 mice (male, 6-8 weeks old) were purchased from Guangdong Yaokang Biotechnology Co., Ltd. (China). According to a common sepsis protocol, we used the cecal ligation and puncture (CLP) method to construct a model of sepsis. For RNA sequencing, 5 mice were randomly divided into 2 groups: the CLP group (n = 3) and the sham group (n = 2). We anesthetized mice in the CLP group with 24% isoflurane. Under aseptic conditions, a 2-cm midline laparotomy was created below the diaphragm to expose the cecum. Two-thirds of the cecum was ligated with a 5-0 silk suture and punctured twice using a 22-gauge needle. The cecum was gently squeezed to extrude a small amount of feces through the perforation site. Animals were resuscitated with 1 mL of subcutaneous saline after CLP. The procedures of the sham group (controls) were the same as the CLP group, except for the ligation and perforation. The mice were sacrificed via neck fracture 6 hours after CLP, and the kidneys were taken for subsequent RNA sequencing.

Thirty specific-pathogen-free (SPF) male C57BL/6 rats at 8 weeks weighing 200-250 g were obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). The rats were housed under room temperatures (25 ± 2 and 12-h light/dark cycle) and given water and food ad libitum before the trial. Then, they were randomly assigned into the control group (n = 10), model group (n = 10), and model + miR-9-5p angomir group (n = 10). All animal procedures were performed under the Care and Use of Laboratory Animals guidelines and approved by the Guangdong Academy of Medical Sciences. According to a previous study, sepsis was induced by the cecal ligation and puncture (CLP) method. Briefly, we anesthetized the rats with 5% chloral hydrate (0.6 ml/100 g body weight) and made a 1.5 cm midline incision on the anterior abdomen to expose the cecum. Then, the cecum was ligated at 30%, punctured twice with a No.4 surgical needle to extrude the fecal content. Finally, 1 ml of normal saline was used for resuscitation. The rats in miR-9-5p angomir group were injected with 100 ng/ul miR-9-5p angomir (RiboBio, Guangzhou, China) into caudal vein (model + miR-9-5p angomir). The rats in the control group experienced the same procedure without ligation and puncture.

Thirty specific-pathogen-free (SPF) male C57BL/6 rats at 8 weeks weighing 200-250 g were obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Briefly, we anesthetized the rats with 5% chloral hydrate (0.6 ml/100 g body weight) and made a 1.5 cm midline incision on the anterior abdomen to expose the cecum. Then, the cecum was ligated at 30%, punctured twice with a No.4 surgical needle to extrude the fecal content. Finally, 1 ml of normal saline was used for resuscitation. The rats in miR-9-5p angomir group were injected with 100 ng/ul miR-9-5p angomir (RiboBio, Guangzhou, China) into caudal vein (model + miR-9-5p angomir). The rats in the control group experienced the same procedure without ligation and puncture.

Thirty specific-pathogen-free (SPF) male C57BL/6 rats at 8 weeks weighing 200-250 g were obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). The rats were housed under room temperatures (25 ± 2 and 12-h light/dark cycle) and given water and food ad libitum before the trial. Then, they were randomly assigned into the control group (n = 10), model group (n = 10), and model + miR-9-5p angomir group (n = 10). All animal procedures were performed under the Care and Use of Laboratory Animals guidelines and approved by the Guangdong Academy of Medical Sciences. According to a previous study, sepsis was induced by the cecal ligation and puncture (CLP) method. Briefly, we anesthetized the rats with 5% chloral hydrate (0.6 ml/100 g body weight) and made a 1.5 cm midline incision on the anterior abdomen to expose the cecum. Then, the cecum was ligated at 30%, punctured twice with a No.4 surgical needle to extrude the fecal content. Finally, 1 ml of normal saline was used for resuscitation. The rats in miR-9-5p angomir group were injected with 100 ng/ul miR-9-5p angomir (RiboBio, Guangzhou, China) into caudal vein (model + miR-9-5p angomir). The rats in the control group experienced the same procedure without ligation and puncture.

Thirty specific-pathogen-free (SPF) male C57BL/6 rats at 8 weeks weighing 200-250 g were obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Briefly, we anesthetized the rats with 5% chloral hydrate (0.6 ml/100 g body weight) and made a 1.5 cm midline incision on the anterior abdomen to expose the cecum. Then, the cecum was ligated at 30%, punctured twice with a No.4 surgical needle to extrude the fecal content. Finally, 1 ml of normal saline was used for resuscitation. The rats in miR-9-5p angomir group were injected with 100 ng/ul miR-9-5p angomir (RiboBio, Guangzhou, China) into caudal vein (model + miR-9-5p angomir). The rats in the control group experienced the same procedure without ligation and puncture.