Six-week-old female BALB/c nude mice were purchased from Byrness Weil Biotechnology Ltd. (Chengdu, China) and housed in a specific pathogen-free environment with a 12-h light/dark cycle and controlled temperature and humidity, and food and water were provided ad libitum. Three million designated treated PLC5 cells were collected and injected subcutaneously into mice. At least 4 mice were used in each group in each experiment. Once the tumours reached a mean volume of 200 mm³, the mice were treated intraperitoneally with sorafenib every 3 days. The mice were then euthanized at the indicated time after injection. Each tumour was dissected, fixed with 4% formaldehyde, and embedded in paraffin. The tumour growth was monitored weekly by calliper measurements.

C57BL/6 mice (female, 6-8 weeks old, 20-30 g weight) and SPF-grade SD rats (female, 180-230 g weight) were used to detect the toxicity of nanoparticles. Different cells (5 x 10⁶) cells were grafted in the left flank; 5 days after engraftation, the stimulated TAMs (1 x 10⁶) were injected into NSG mice through the tail vein. Different treatments were given and recorded as day 0.

Adult male Sprague-Dawley rats weighing 200-220 g were purchased from the Animal Center of the Medical Department of Xi'an Jiaotong University. The rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg), and the spines of the rats were fixed. The skin of T9-12 level was incised, and laminectomy was performed at the T10 level to expose the spinal cord. The rats were then placed in the appropriate position of the impactor so that 10 g of rod fell freely at a height of 3 cm and hit the center of the T10 level of the spinal cord. The signs of successful establishment of the model were the appearance of hind limb extension and tail-flick reflex in rats. Laminectomy was performed only in the sham-operated group. The rats in the SCI group received an artificial bladder massage twice a day to assist in urination until they were able to urinate.

BALB/c nude mice (male, 4 weeks old) were purchased from the Animal Center of Nanjing University with free access to water and food. A549 cells (10⁶ cells per mouse) transfected with miR-4443 mimic or mimic-NC were injected subcutaneously to generate subcutaneous tumors. Tumor volume was recorded.

Male C57BL/6 (7-8 weeks) were purchased from the Animal Center of Nanjing University (Nanjing, China) and housed in the condition with controlled temperature and humidity under a 12-h light/dark circadian rhythm. For determining the effects of MSCs-exo, the animals were derived into three groups: sham (n = 10), ASCI (n = 10), ASCI + MSCs (n = 10); for determining the effects of exosomal lncGm36569, the animals were divided into five groups: sham (n = 10), ASCI (n = 10), ASCI + MSC-Exo (ctrl) (n = 10), ASCI + MSCs-Exo (lnc-OE) (n =10), ASCI + MSCs-Exo (si-lnc) (n = 10).

C57BL/6 mice (female, 6-8 weeks old, 20-30 g weight) and SPF-grade SD rats (female, 180-230 g weight) were used to detect the toxicity of nanoparticles. Different cells (5 x 10⁶) cells were grafted in the left flank; 5 days after engraftation, the stimulated TAMs (1 x 10⁶) were injected into NSG mice through the tail vein. Different treatments were given and recorded as day 0.

Male C57BL/6 (7-8 weeks) were purchased from the Animal Center of Nanjing University (Nanjing, China) and housed in the condition with controlled temperature and humidity under a 12-h light/dark circadian rhythm. For determining the effects of MSCs-exo, the animals were derived into three groups: sham (n = 10), ASCI (n = 10), ASCI + MSCs (n = 10); for determining the effects of exosomal lncGm36569, the animals were divided into five groups: sham (n = 10), ASCI (n = 10), ASCI + MSC-Exo (ctrl) (n = 10), ASCI + MSCs-Exo (lnc-OE) (n =10), ASCI + MSCs-Exo (si-lnc) (n = 10).

Six-week-old female BALB/c nude mice were purchased from Byrness Weil Biotechnology Ltd. (Chengdu, China) and housed in a specific pathogen-free environment with a 12-h light/dark cycle and controlled temperature and humidity, and food and water were provided ad libitum. Three million designated treated PLC5 cells were collected and injected subcutaneously into mice. At least 4 mice were used in each group in each experiment. Once the tumours reached a mean volume of 200 mm³, the mice were treated intraperitoneally with sorafenib every 3 days. The mice were then euthanized at the indicated time after injection. Each tumour was dissected, fixed with 4% formaldehyde, and embedded in paraffin. The tumour growth was monitored weekly by calliper measurements.

BALB/c nude mice (male, 4 weeks old) were purchased from the Animal Center of Nanjing University with free access to water and food. A549 cells (10⁶ cells per mouse) transfected with miR-4443 mimic or mimic-NC were injected subcutaneously to generate subcutaneous tumors. Tumor volume was recorded.

Female BALB/c nude mice aged 4 weeks were administered subcutaneously with 2 x 10⁶ cells (five mice/group). After that, the mice were given intratumoural inoculation of 40 uL si-circGFRA1 or si-NC every 4 days. To detect lung metastases, we intravenously inoculated 1 x 10⁵ cells into the tail veins of mice (six mice/group). After the elapse of 8 weeks, we anaesthetized the mice, harvested their lungs and visually counted the metastatic nodules in the lungs, followed by validation via H&E staining and counting under a microscope.

Female BALB/c nude mice aged 4 weeks were administered subcutaneously with 2 x 10⁶ cells (five mice/group). After that, the mice were given intratumoural inoculation of 40 uL si-circGFRA1 or si-NC every 4 days. To detect lung metastases, we intravenously inoculated 1 x 10⁵ cells into the tail veins of mice (six mice/group). After the elapse of 8 weeks, we anaesthetized the mice, harvested their lungs and visually counted the metastatic nodules in the lungs, followed by validation via H&E staining and counting under a microscope.

Briefly, surgically resected tumors were maintained in DMEM-F12 (Gibco) supplemented with 1% HEPES (Sigma-Aldrich), 1% L-glutamine (Gibco), 10% FBS (Gibco), 2% penicillin/streptomycin (Sigma-Aldrich), and 10 uM Y-27632 (R&D Systems). Tumors were digested with collagenase solution (5 mL of the above medium with 75 uL collagenase, 124 ug/mL dispase type II, and 0.2% Primocen) for 30 min and then filtered through a 70 um filter (Corning). An organoid pellet was obtained by centrifugation (200x g for 10 min). Organoids were suspended in Matrigel (Corning, Tehama County, CA) with IntestiCult Organoid Growth Medium (#06010, STEMCELL Technologies) and seeded in 12-well plates. Approximately 750 uL of IntestiCult Organoid Growth Medium was added to each well. Organoids were divided into five groups of control, curcumin (3.0 ug/mL), andrographis (30.0 ug/mL), their combination (curcumin; 3.0 ug/mL, andrographis; 30.0 ug/mL), and their combination plus ferrostatin-1 (curcumin; 3.0 ug/mL, andrographis; 30.0 ug/mL; ferrostatin-1; 20 uM). Following forty-eight hours of treatment, the numbers of organoids (<100 um) and their mean sizes were examined using Image J software.

Briefly, surgically resected tumors were maintained in DMEM-F12 (Gibco) supplemented with 1% HEPES (Sigma-Aldrich), 1% L-glutamine (Gibco), 10% FBS (Gibco), 2% penicillin/streptomycin (Sigma-Aldrich), and 10 uM Y-27632 (R&D Systems). Tumors were digested with collagenase solution (5 mL of the above medium with 75 uL collagenase, 124 ug/mL dispase type II, and 0.2% Primocen) for 30 min and then filtered through a 70 um filter (Corning). An organoid pellet was obtained by centrifugation (200x g for 10 min). Organoids were suspended in Matrigel (Corning, Tehama County, CA) with IntestiCult Organoid Growth Medium (#06010, STEMCELL Technologies) and seeded in 12-well plates. Approximately 750 uL of IntestiCult Organoid Growth Medium was added to each well. Organoids were divided into five groups of control, curcumin (3.0 ug/mL), andrographis (30.0 ug/mL), their combination (curcumin; 3.0 ug/mL, andrographis; 30.0 ug/mL), and their combination plus ferrostatin-1 (curcumin; 3.0 ug/mL, andrographis; 30.0 ug/mL; ferrostatin-1; 20 uM). Following forty-eight hours of treatment, the numbers of organoids (<100 um) and their mean sizes were examined using Image J software.

Adult Sprague-Dawley rats (70 days old) of both sexes were purchased from the Laboratory Animal Centre of Harbin Medical University, Harbin, China. Before the experiment, female rats were allowed to acclimatize for a minimum of 1 week and then were monitored daily by vaginal lavage to determine the stage of the estrous cycle as previously described (Zhanget al., 2016). Pregnancy was achieved by housing female rats on the night of proestrus with fertile males of the same strain at a 2:1 ratio. Confirmation of mating was performed the morning after by the presence of a vaginal plug, and this was considered as GD 0.5. The rats were sacrificed between 8:00 a.m. and 9:00 a.m. hours on GD 14.5.

Fifty male C57BL/6 mice provided by Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China) resided in an animal house (temperature 26 ± 1 , humidity 50 ± 10%), in which the light was on for 12 h and off for 12 h. Mice were acclimatised for 1 week and allowed water and animal food with no limitations. Then, all mice were stochastically divided into 5 groups using random number tables available online (https://www.random-online.com/, accessed on 26 December 2021), including: (1) C group, a control group treated with normal saline for 7 consecutive days (n = 10); (2) M group, a model group with intraperitoneal injection of 20 mg/kg/day MPTP (Sigma-Aldrich, Taufkirchen, Germany, M0896) for 7 consecutive days (n = 10); (3) L group, treated with MPTP and 0.4 mg/kg/day liraglutide for 7 consecutive days (n = 10); (4) R group, treated with MPTP and 109 colony-forming unit (CFU) L. lactis MG1363 for 7 consecutive days via gavage (n = 10); (5) RG group, treated with MPTP and 109 CFUL. lactis MG1363-pMG36e-GLP-1 for 7 consecutive days via gavage (n = 10). All animals survived treatment and all animal experiments were administered from 9:00 to 12:00 in the morning to reduce systematic errors.

We purchased forty-eight 3-week-old male C57BL/6N mice from Beijing HFK Bioscience Co. Ltd. and gave a twelve-hour light and dark cycle starting from 06:00 (am) to 18:00 (pm). Mice were randomly assigned into three groups after 2 weeks of adaptive feeding as follows. (1) The control group (n = 16): mice were provided with normal drinking water, a normal diet and intraperitoneal administration of solvent (5% DMSO + 40% Peg300 + 5% Tween 80 + 50% ddH2O) 3 mL/kg once a week aged 13 to 17 weeks. (2) The HFpEF group (n = 16): a double-hit model was designed, in which metabolic and mechanical stress worked together and resulted in HFpEF. Briefly, C57BL/6N mice had unrestricted access to a high-fat diet (HFD, D12492, Research Diet) starting from 5 weeks old. Meanwhile, a nitric oxide synthase inhibitor, N (gamma)-nitro-L-arginine methyl ester (L-NAME) (N5751, Sigma) was supplied in drinking water (0.5 g/L) for HFpEF groups, and the pH of the drinking water was adjusted to 7.4. The above placebo solvent was administrated in the same manner. (3) The HFpEF + Levo group (n = 16): according to the previous study, HFpEF mice received 3 mg/kg levosimendan (S2446, Selleck) (Dissolve 1 mg of levosimendan in 50 uL of DMSO, subsequently dilute to 1 mg/mL with the above solvent) intraperitoneally once a week from week 13 to 17.

C57BL/6 J mice (8-week old) from SLAC Laboratory Animal Co. LTD (Shanghai, China) were housed in a specific pathogen-free (SPF) barrier system at 20 with 12-h light/dark cycles. They were randomly grouped as follows: (1) intratracheal saline (control group); (2) intraperitoneal deferoxamine (DFO, Sigma-Aldrich; DFO group); (3) intratracheal bleomycin (BLM, Nippon Kayaku Co., Ltd.; BLM group); and (4) intratracheal BLM plus intraperitoneal deferoxamine (BLM + DFO group). They were intratracheally injected with 50 ul of BLM (5 mg/kg) on day 0. For the preventive anti-fibrotic treatment, DFO (50 mg/kg2 day-1) was administered from day 0 to day 20. Lung samples were collected at day 21.