OBJECTIVES: To study the effect of gut microbiota on hematopoiesis in a neonatal rat model of necrotizing enterocolitis (NEC). METHODS: Neonatal Sprague-Dawley rats were randomly divided into a control group and a model group (NEC group), with 6 rats in each group. Formula milk combined with hypoxia and cold stimulation was used to establish a neonatal rat model of NEC. Hematoxylin and eosin staining was used to observe the pathological changes of intestinal tissue and hematopoiesis-related organs. Routine blood tests were conducted for each group. Immunohistochemistry was used to observe the changes in specific cells in hematopoiesis-related organs. Flow cytometry was used to measure the changes in specific cells in bone marrow. 16S rDNA sequencing was used to observe the composition and abundance of gut microbiota. RESULTS: Compared with the control group, the NEC group had intestinal congestion and necrosis, damage, atrophy, and shedding of intestinal villi, and a significant increase in NEC histological score. Compared with the control group, the NEC group had significantly lower numbers of peripheral blood leukocytes and lymphocytes (P<0.05), nucleated cells in the spleen, thymus, and bone marrow, and small cell aggregates with basophilic nuclei in the liver (P<0.05). The NEC group had significant reductions in CD71⁺ erythroid progenitor cells in the liver, CD45⁺ lymphocytes in the spleen and bone marrow, CD3⁺ T lymphocytes in thymus, and the proportion of CD45⁺CD3^-CD43⁺SSC^hi neutrophils in bone marrow (P<0.05). There was a significant difference in the composition of gut microbiota between the NEC and control groups, and the NEC group had a significant reduction in the abundance of Ligilactobacillus and a significant increase in the abundance of Escherichia-Shigella (P<0.05), which replaced Ligilactobacillus and became the dominant flora. CONCLUSIONS Multi-lineage hematopoietic disorder may be observed in a neonatal rat model of NEC, which may be associated with gut microbiota dysbiosis and abnormal multiplication of the pathogenic bacteria Escherichia-Shigella.
Rats ; Animals ; Enterocolitis, Necrotizing/etiology* ; Gastrointestinal Microbiome ; Rats, Sprague-Dawley ; Animals, Newborn ; Infant, Newborn, Diseases

Objective: To investigate the poisonous substances and geographical distribution of poisoning in children in China. Methods: A cross-sectional study. The clinical data of 8 385 hospitalized children from January 2016 to December 2020 were extracted from the FUTang Updating Medical Records database. These children aged 0 to 18 years and were admitted due to poisoning. They were grouped according to age (newborns and infants, toddlers, preschoolers, school-age children, adolescents), place of residence (Northeast China, North China, Central China, East China, South China, Southwest China, Northwest China), and mode of discharge (discharge under medical advice, transfer to another hospital under medical advice, discharge without medical advice, death, other). The poisonous substance and causes of poisoning in different groups were analyzed. Results: Among these 8 385 children, 4 734 (56.5%) were male and 3 651 (43.5%) female, with a male-to-female ratio of 1.3∶1. The age was 3 (2, 7) years. The prevalence of poisoning was 51.8% (4 343/8 385) in toddlers, 16.5% (1 380/8 385) in adolescents, 14.8% (1 242/8 385) in preschoolers, 14.4% (1 206/8 385) in school-age children, and 2.5% (214/8 385) in newborns and infants. Drug poisoning accounted for 43.5% (3 649/8 385) and pesticide accounted for 26.8% (2 249/8 385). Drug poisoning was more common in adolescents (684/1 380, 49.6%) and toddlers (2 041/4 343, 47.0%); non-drug poisoning was more common in school-age children (891/1 206, 73.9%), of which carbon monoxide was mainly in newborns and infants (41/214, 19.2%) and food poisoning in children of school age (241/1 206, 20.0%). Regarding regional characteristics, drug poisoning was more frequent in South China (188/246, 64.2%) and non-drug poisoning was more frequent in Southwest China (815/1 123, 72.5%). For drugs, anti-epileptic drugs, sedative-hypnotic drugs and anti-Parkinson's disease drugs had a higher proportion of poisoning in North China (138/1 034, 13.0%) than that in other regions. For non-drug poisoning, pesticides (375/1 123, 33.3%), food poisoning (209/1 123, 18.6%) and contact with poisonous animals (86/1 123, 7.7%) were more common in Southwest China than in other regions; carbon monoxide poisoning was more common in North China (81/1 034, 7.6%) and Northwest China (65/1 064, 6.3%). In Central China, poisoning happened more in toddlers (792/1 295, 61.2%) and less in adolescents (115/1 295, 8.8%) than in other regions. Regarding different age groups, poisoning in adolescent happened more in Northeast China (121/457, 26.5%), North China (240/1 034, 23.2%), and Northwest China (245/1 064, 23.0%). The rate of discharge under medical advice, discharge without medical advice, and mortality rate within the 5 years were 77.0% (6 458/8 385), 20.8% (1 743/8 385), 0.5% (40/8 385), respectively. Conclusions: Poisoning is more common in male and toddlers. Poisonous substances show a regional characteristic and vary in different age groups, with drugs and insecticides as the most common substances.
Infant ; Adolescent ; Animals ; Child ; Male ; Humans ; Infant, Newborn ; Female ; Child, Hospitalized ; Cross-Sectional Studies ; Carbon Monoxide Poisoning/epidemiology* ; Pesticides ; Foodborne Diseases ; Hospitals ; Drug-Related Side Effects and Adverse Reactions ; China/epidemiology*

Objective To investigate the protective effect of artesunate on hypoxic-ischemic brain damage (HIBD) and its mechanism in neonatal rats. Methods 7-day-old neonatal SD rats were randomly divided into sham operation group, model group, artesunate 5 mg/kg group, artesunate 10 mg/kg group, artesunate 20 mg/kg group and dexamethasone 6 mg/kg group, with 18 rats in each group. HIBD models were established in groups except for the sham operation group. The sham operation group only needed to separate the left common carotid artery without ligation and nitrogen-oxygen mixed gas ventilation. Each group was injected with drug intraperitoneally right after surgery and the rats in the sham operation group and the model group were injected with an equal volume of normal saline (once a day for a total of 5 times). One hour after the last injection, the rats in each group were scored for neurological defects. After the rats were sacrificed, the brain water content was measured and the pathological changes of the brain tissues of rats were observed. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) was used to detect the neuronal cell apoptosis, and ELISA was applied to detect the levels of IL-1β, IL-6 and TNF-α in brain tissues and peripheral blood of each group of rats. Western blot analysis was adopted to detect the protein expression levels of NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1 in the rats brain tissues of each group. Results Compared with the model group, the neurological deficit score was decreased; the pathological damage of brain tissues was relieved; the brain water content was significantly reduced; the apoptosis number of hippocampal neurons was decreased significantly; the levels of IL-1β, IL-6 and TNF-α in brain tissues and peripheral blood were significantly reduced; the protein expression levels of NLRP3, ASC and caspase-1 were significantly lowered in the middle-dose and high-dose artesunate groups and the dexamethasone group. Conclusion Artesunate can improve the neurological function, relieve the brain damage, and alleviate the brain edema in neonatal rats with HIBD. It can protect the HIBD, which may be related to the inhibition of NLRP3 inflammasome activation and reduction of inflammatory cytokine secretion.
Animals ; Rats ; Animals, Newborn ; Artesunate/pharmacology* ; Brain/metabolism* ; Caspases/metabolism* ; Dexamethasone ; Hypoxia-Ischemia, Brain/pathology* ; Inflammasomes ; Interleukin-6/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha/metabolism* ; Water/metabolism*

OBJECTIVES: To study the effect of platelet-derived growth factor-BB (PDGF-BB) on pulmonary vascular remodeling in neonatal rats with hypoxic pulmonary hypertension (HPH). METHODS: A total of 128 neonatal rats were randomly divided into four groups: PDGF-BB+HPH, HPH, PDGF-BB+normal oxygen, and normal oxygen (n=32 each). The rats in the PDGF-BB+HPH and PDGF-BB+normal oxygen groups were given an injection of 13 μL 6×10¹⁰ PFU/mL adenovirus with PDGF-BB genevia the caudal vein. After 24 hours of adenovirus transfection, the rats in the HPH and PDGF-BB+HPH groups were used to establish a neonatal rat model of HPH. Right ventricular systolic pressure (RVSP) was measured on days 3, 7, 14, and 21 of hypoxia. Hematoxylin-eosin staining was used to observe pulmonary vascular morphological changes under an optical microscope, and vascular remodeling parameters (MA% and MT%) were also measured. Immunohistochemistry was used to measure the expression levels of PDGF-BB and proliferating cell nuclear antigen (PCNA) in lung tissue. RESULTS: The rats in the PDGF-BB+HPH and HPH groups had a significantly higher RVSP than those of the same age in the normal oxygen group at each time point (P<0.05). The rats in the PDGF-BB+HPH group showed vascular remodeling on day 3 of hypoxia, while those in the HPH showed vascular remodeling on day 7 of hypoxia. On day 3 of hypoxia, the PDGF-BB+HPH group had significantly higher MA% and MT% than the HPH, PDGF-BB+normal oxygen, and normal oxygen groups (P<0.05). On days 7, 14, and 21 of hypoxia, the PDGF-BB+HPH and HPH groups had significantly higher MA% and MT% than the PDGF-BB+normal oxygen and normal oxygen groups (P<0.05). The PDGF-BB+HPH and HPH groups had significantly higher expression levels of PDGF-BB and PCNA than the normal oxygen group at all time points (P<0.05). On days 3, 7, and 14 of hypoxia, the PDGF-BB+HPH group had significantly higher expression levels of PDGF-BB and PCNA than the HPH group (P<0.05), while the PDGF-BB+normal oxygen group had significantly higher expression levels of PDGF-BB and PCNA than the normal oxygen group (P<0.05). CONCLUSIONS Exogenous administration of PDGF-BB in neonatal rats with HPH may upregulate the expression of PCNA, promote pulmonary vascular remodeling, and increase pulmonary artery pressure.
Rats ; Animals ; Hypertension, Pulmonary ; Becaplermin ; Animals, Newborn ; Proliferating Cell Nuclear Antigen ; Vascular Remodeling ; Pulmonary Artery/metabolism* ; Hypoxia ; Oxygen ; Cell Proliferation ; Myocytes, Smooth Muscle/metabolism*

OBJECTIVES: To study the effect of ligustrazine injection on mitophagy in neonatal rats with hypoxic-ischemic encephalopathy (HIE) and its molecular mechanism. METHODS: Neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group with 8 rats, a model group with 12 rats, and a ligustrazine group with 12 rats. The rats in the model group and the ligustrazine group were used to establish a neonatal rat model of HIE by ligation of the left common carotid artery followed by hypoxia treatment, and blood vessels were exposed without any other treatment for the rats in the sham-operation group. The rats in the ligustrazine group were intraperitoneally injected with ligustrazine (20 mg/kg) daily after hypoxia-ischemia, and those in the sham-operation group and the model group were intraperitoneally injected with an equal volume of normal saline daily. Samples were collected after 7 days of treatment. Hematoxylin and eosin staining and Nissl staining were used to observe the pathological changes of neurons in brain tissue; immunohistochemical staining was used to observe the positive expression of PINK1 and Parkin in the hippocampus and cortex; TUNEL staining was used to measure neuronal apoptosis; Western blotting was used to measure the expression levels of the mitophagy pathway proteins PINK1 and Parkin and the autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and ubiquitin-binding protein (P62). RESULTS: Compared with the sham-operation group, the model group had a significant reduction in the number of neurons, an increase in intercellular space, loose arrangement, lipid vacuolization, and a reduction in Nissl bodies. The increased positive expression of PINK1 and Parkin, apoptosis rate of neurons, and protein expression levels of PINK1, Parkin, Beclin1 and LC3 (P<0.05) and the decreased protein expression level of P62 in the hippocampus were also observed in the model group (P<0.05). Compared with the model group, the ligustrazine group had a significant increase in the number of neurons with ordered arrangement and an increase in Nissl bodies, significant reductions in the positive expression of PINK1 and Parkin, the apoptosis rate of neurons, and the protein expression levels of PINK1, Parkin, Beclin1, and LC3 (P<0.05), and a significant increase in the protein expression level of P62 (P<0.05). CONCLUSIONS Ligustrazine can alleviate hypoxic-ischemic brain damage and inhibit neuronal apoptosis in neonatal rats to a certain extent, possibly by inhibiting PINK1/Parkin-mediated autophagy.
Rats ; Animals ; Hypoxia-Ischemia, Brain/metabolism* ; Animals, Newborn ; Rats, Sprague-Dawley ; Beclin-1 ; Autophagy ; Ubiquitin-Protein Ligases/metabolism* ; Protein Kinases/metabolism*

OBJECTIVE: To investigate the effect of palmitic acid (PA) on autophagy in neonatal rat cardiomyocytes (NRCMs) and explore the underlying mechanism. METHODS: NRCMs were isolated and cultured for 24 h before exposure to 10% BSA and 0.1, 0.3, 0.5, or 0.7 mmol/L PA for 24 h. After the treatments, the expressions of Parkin, PINK1, p62, LC3Ⅱ/ LC3Ⅰ, cGAS, STING and p-IRF3/IRF3 were detected using Western blotting and the cell viability was assessed with CCK8 assay, based on which 0.7 mmol/L was selected as the optimal concentration in subsequent experiments. The effects of cGAS knockdown mediated by cGAS siRNA in the presence of PA on autophagy-related proteins in the NRCMs were determined using Western blotting, and the expressions of P62 and LC3 in the treated cells were examined using immunofluorescence assay. RESULTS: PA at different concentrations significantly lowered the expressions of Parkin, PINK1, LC3 Ⅱ/LC3 Ⅰ and LC3 Ⅱ/LC3 Ⅰ+Ⅱ (P < 0.05), increased the expression of p62 (P < 0.05), and inhibited the viability of NRCMs (P < 0.05). Knockdown of cGAS obviously blocked the autophagy-suppressing effect of PA and improved the viability of NRCMs (P < 0.05). CONCLUSION PA inhibits autophagy by activating the cGAS-STING-IRF3 pathway to reduce the viability of NRCMs.
Animals ; Animals, Newborn ; Autophagy ; Myocytes, Cardiac ; Nucleotidyltransferases/pharmacology* ; Palmitic Acid/pharmacology* ; Rats

Folic acid belongs to the group of water-soluble B vitamins and naturally exists in multiple forms in a wide variety of foods such as legumes, vegetables, liver, and milk (Iyer and Tomar, 2009; Lyon et al., 2020). It is involved in many biochemical reactions critical for cell division, such as purine and pyrimidine biosynthesis, DNA/RNA biosynthesis, and amino acid metabolism (Iyer and Tomar, 2009). Mammals cannot synthesize folic acid and thus they must acquire it from food. Although folic acid is ubiquitous in foods, folic acid deficiency still often occurs due to various causes such as unhealthy diet (Hildebrand et al., 2021; Iimura et al., 2022), disease-related malabsorption (Arcot and Shrestha, 2005), medication-related depletion (Arcot and Shrestha, 2005), or vitamin B12 deficiency (Fishman et al., 2000). Folic acid deficiency has been associated with several health problems, such as anemia (Carmel, 2005; Bailey and Caudill, 2012), cancer (Duthie, 1999), cardiovascular diseases (Wald et al., 2002), neural tube defects in newborns (van der Put et al., 2001), neuropsychiatric dysfunction (Shea et al., 2002), depression (Falade et al., 2021), inflammatory diseases (Suzuki and Kunisawa, 2015; Jones et al., 2019), and eye diseases (Sijilmassi, 2019). To prevent folic acid deficiency, its daily intake (400 μg/d) has been recommended for adults in the European Union, and its increased intake (600 μg/d) is advised for women before and during pregnancy (FAO/WHO, 2002; IOM, 2004). The New Zealand government mandated the fortification of non-organic wheat flour with folic acid in July 2021, and the UK government mandated the fortification of non-wholemeal wheat flour with folic acid in September 2021 (Haggarty, 2021).
Adult ; Animals ; Female ; Flour ; Folic Acid/metabolism* ; Folic Acid Deficiency/prevention & control* ; Food, Fortified ; Humans ; Infant, Newborn ; Mammals/metabolism* ; Pregnancy ; Triticum/metabolism*

Our previous studies have shown that calcitonin gene-related peptide (CGRP) exerts protective effects on the acute lung injury induced by oxidative stress. This study was aimed to investigate whether autophagy was involved in the protection of CGRP against oxidative stress-induced lung injury in neonatal rats. Newborn Sprague-Dawley (SD) rats were randomly divided into five groups: Control group, oxidative stress model group (Model group), Model + CGRP group, Model + CGRP + Rapamycin (an autophagy agonist) group, and Model + CGRP + LY294002 (an autophagy inhibitor) group. The model of hyperoxia-induced lung injury was established by continuous inhalation of oxygen (FiO₂ = 90%-95%) for 14 days in neonatal SD rats. Pathological changes of lung tissue were observed by hematoxylin and eosin (HE) staining, and mean linear intercept (MLI) was measured. The quantitative changes of autophagic vesicles (AV) in type II alveolar epithelial cells (AECII) were measured under the transmission electron microscope. The protein expressions of Caspase-3, Bcl-2, mTOR, and Beclin-1 in lung tissue lysates were detected by Western blot. The results showed that, compared to the Model group at the same time point, the number of AV in AECII and the expression level of Beclin-1 protein of the lung tissue were increased, while the expression level of mTOR protein was decreased, with alleviated pathological changes, reduced MLI value and Caspase-3 protein expression level, increased Bcl-2 protein expression level in the lung tissue of Model + CGRP group. In addition, we found that the protective effect of CGRP on hyperoxia-induced lung injury could be enhanced by autophagy activator Rapamycin and abolished by autophagy inhibitor LY294002. Together, these findings indicate that CGRP could attenuate hyperoxia-induced lung injury in neonatal rats by enhancing autophagy.
Acute Lung Injury/pathology* ; Animals ; Animals, Newborn ; Autophagy ; Calcitonin/metabolism* ; Calcitonin Gene-Related Peptide/metabolism* ; Caspase 3/metabolism* ; Hyperoxia/pathology* ; Lung/pathology* ; Lung Injury/prevention & control* ; Oxidative Stress ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Rats ; Rats, Sprague-Dawley ; Sirolimus/pharmacology*

OBJECTIVE: To construct an adenovirus vector expressing artificial splicing factor capable of regulating alternative splicing of Yap1 in cardiomyocytes. METHODS: The splicing factors with different sequences were constructed against Exon6 of YAP1 based on the sequence specificity of Pumilio1. The PCR fragment of the artificially synthesized PUF-SR or wild-type PUFSR was cloned into pAd-Track plasmid, and the recombinant plasmids were transformed into E. coli DH5α for plasmid amplification. The amplified plasmids were digested with Pac I and transfected into 293A cells for packaging to obtain the adenovirus vectors. Cultured neonatal rat cardiomyocytes were transfected with the adenoviral vectors, and alternative splicing of YAP1 was detected using quantitative and semi-quantitative PCR; Western blotting was performed to detect the signal of the fusion protein Flag. RESULTS: The transfection efficiency of the adenovirus vectors was close to 100% in rat cardiomyocytes, and no fluorescent protein was detected in the cells with plasmid transfection. The results of Western blotting showed that both the negative control and Flag-SR-NLS-PUF targeting the YAPExon6XULIE sequence were capable of detecting the expression of the protein fused to Flag. The results of reverse transcription-PCR and PCR demonstrated that the artificial splicing factor constructed based on the 4th target sequence of YAP1 effectively regulated the splicing of YAP1 Exon6 in the cardiomyocytes (P < 0.05). CONCLUSION We successfully constructed adenovirus vectors capable of regulating YAP1 alternative splicing rat cardiomyocytes.
Adenoviridae/metabolism* ; Alternative Splicing ; Animals ; Animals, Newborn ; Escherichia coli/metabolism* ; Genetic Vectors ; Myocytes, Cardiac/metabolism* ; Plasmids ; RNA Splicing Factors/metabolism* ; Rats ; Transfection

Neuroinflammation is a key contributor to the pathogenic cascades induced by hypoxic-ischemic (HI) insult in the neonatal brain. AD-16 is a novel anti-inflammatory compound, recently found to exert potent inhibition of the lipopolysaccharide-induced production of pro-inflammatory and neurotoxic mediators. In this study, we evaluated the effect of AD-16 on primary astrocytes and neurons under oxygen-glucose deprivation (OGD) in vitro and in mice with neonatal HI brain injury in vivo. We demonstrated that AD-16 protected against OGD-induced astrocytic and neuronal cell injury. Single dose post-treatment with AD-16 (1 mg/kg) improved the neurobehavioral outcome and reduced the infarct volume with a therapeutic window of up to 6 h. Chronic administration reduced the mortality rate and preserved whole-brain morphology following neonatal HI. The in vitro and in vivo effects suggest that AD-16 offers promising therapeutic efficacy in attenuating the progression of HI brain injury and protecting against the associated mortality and morbidity.
Animals ; Animals, Newborn ; Astrocytes/pathology* ; Brain/pathology* ; Brain Injuries/pathology* ; Glucose ; Hypoxia ; Hypoxia-Ischemia, Brain/drug therapy* ; Mice ; Neuroinflammatory Diseases ; Neuroprotective Agents/therapeutic use* ; Oxygen/therapeutic use*