HIBD is a common disease found in newborns which often presents as a series of neurological sequelae. Acupuncture, as a therapy of alternative medicine, has achieved a remarkable curative effect in the treatment of this disease 20,21. Meanwhile, several studies have documented the important neuroprotective roles of acupuncture in cerebral functional compensation in animal models with HIBD 22-24. With the rise of RNA-seq technology, people gradually became interested in the gene-related mechanism of HIBD and began to do research 25,26. However, the role of acupuncture in regulating RNAs in HIBD rats remains unknown.
In this study, HIBD rats showed significantly longer new object recognition times, suggesting that HIBD rats may be worse than normal rats in terms of learning and memory. This result is consistent with a number of studies indicated that HIBD rats perform poorly in behavioral tests of learning and memory abilities, such as water maze, shuttle box, T maze, and radial maze 27-29. As a common neonatal nervous system disease, HIBD can lead to different degrees of brain damage, such as brain metabolism disorder, neuronal apoptosis, reduced brain flow, or even cerebral hemorrhage in severe cases, resulting in liquefaction and necrosis of brain tissue 30-32. Our results were in line with these findings in that we found that there were significantly fewer Nissl corpuscles in the prefrontal lobe of HIBD rats, indicating that HIBD can indeed cause damage to neurons in the brains of rats. Acupuncture, as an alternative therapy, is attracting increasing attention for its ideal curative effect. Recently, it has been reported that acupuncture can improve locomotor activity and learning–memory ability by improving hippocampal cellular autophagy in rats with HIBD, suggesting that acupuncture could effectively alleviate the brain injury caused by HIBD 33. Additionally, Yuan Q et al. found that acupuncture could significantly reduce the expression of CYT-C and caspase-3 in the cerebral cortex of HIBD rats and reduce the apoptosis of neurons to protect the brain tissue 11. Consistent with these results, we found that the number of Neisseria bodies in HIBD rats treated with acupuncture was significantly higher than that in untreated rats. Additionally, the performance of the new object recognition experiment of the rats after acupuncture was better than that of the untreated HIBD rats, indicating that acupuncture is an effective therapy for HIBD.
In order to explore the target of acupuncture, we identified RNA differentially expressed in the PFC of three groups of rats and found something interesting: 1) lncRNAs had fewer exons, shorter lengths, and shorter lengths of the ORF than mRNAs; 2) from the gene heat map, we found that the effect of acupuncture intervention on lncRNA was more obvious than that of mRNA and circRNA. Moreover, the Venn diagram showed that the number of genes in the overlap part of lncRNA was higher than that of mRNA and circRNA; 3) 877 differentially expressed lncRNAs were identified, 65 lncRNAs were significantly and differentially expressed in acupuncture rats relative to model rats with 17 up-regulated and 48 down-regulated, and 410 lncRNAs were significantly and differentially expressed in the model rats relative to the normal rats with 372 up-regulated and 38 down-regulated; 4) the GO analysis showed that lncRNAs that were differentially expressed between the acupuncture and model groups were involved in neurological system processes, plasma membrane, olfactory receptor activity, and receptor activity, whereas the lncRNAs that were differentially expressed between the model and normal groups were involved in olfactory receptor activity, G-protein-coupled receptor activity, and sensory perception. The KEGG pathway analysis revealed that lncRNAs that were differentially expressed between the acupuncture and model groups participated in multiple pathways, including olfactory transduction and ribosomes, cytokine–cytokine receptor interactions, and taste transduction. In the comparison between the model and normal groups, the differentially expressed lncRNAs were associated with pathways, such as olfactory transduction, ribosomes, drug metabolism and other enzymes, and biosynthesis of amino acids; and 5) 30 circRNAs, 47 lncRNAs, and 11 mRNAs with differential expression shared a common binding site for miRNA between the acupuncture and model groups. Eighty-eight circRNAs, 162 lncRNAs, and 30 mRNAs with differential expression shared a common binding site for miRNA between the model and normal groups.
TCONS_00013568 was an up-regulated lncRNA in the model group but was decreased in the acupuncture group. Through KEGG pathway analysis, we found that the target gene of TCONS_00013568, Epo, was related to the Jak-STAT signaling pathway, HIF-1 signaling pathway, and PI3K-Akt signaling pathway. Reportedly, the inhibition of the JAK/STAT pathway can not only promote brain-derived neurotrophic factors and hippocampal neuron proliferation to alleviate autism symptoms 34 but also protect against α-synuclein-induced neuroinflammation and dopaminergic neurodegeneration 35. Some researchers have found that the improvement in EPO was associated with the neuroprotective effect of the JAK/STAT pathway in cerebral palsy 36. The HIF-1 signaling pathway was found to be associated with stroke, vascular dementia, and chronic hypoxia 37-39. Activating the HIF-1 signaling pathway could exert angiogenic and anti-apoptotic effects against cerebral ischemia-reperfusion injury 40. Hippocampal neurons could be protected against excessive autophagy and apoptosis through the PI3K/Akt signaling pathway 41. EPO was found to promote cerebrovascular regeneration by activating the PI3K/Akt signaling pathway in premature brain damage 42. In our results, Cdkn2a, the other target gene of TCONS_00013568, was also related to glioma and the p53 signaling pathway. The results of several studies showed that the p53 signaling pathway was closely related to Alzheimer’s disease, glioblastoma multiforme, and ischemic stroke 43-45. It has been reported that Cdkn2a plays an important role in apoptosis by activating the p53 signaling pathway 46. The new target genes of TCONS_00013568 in the above pathways may provide new therapeutic targets for the treatment of HIBD. However, the detailed mechanism needs further study.
TCONS_00068680 was another up-regulated lncRNA in the model group with down-regulation in the acupuncture group. Several target genes of TCONS_00068680 participated in the Jak-STAT signaling pathway, such as Il9r and Csf3. The expressed protein of Il9r is the receptor of interleukin (IL)-9. Some researchers found that IL-9 was important in driving immune responses to chronic inflammation and that the blockade of IL-9 signaling in the Jak-STAT signaling pathway may be novel therapeutic targets for inflammatory diseases in the mucosal immune system 47. Furthermore, IL-9 is closely associated with multiple sclerosis and could reduce inflammation and provide protection from neurodegeneration 48. The expressed protein of Csf3 is a potent activator of neutrophil ROS, which is related to stroke. It was found to be neuroprotective in acute and chronic settings through various mechanisms, including the enhancement of neurogenesis and angiogenesis, as well as the apoptosis and suppression of inflammation 49. Moreover, Csf3 was involved in the PI3K-Akt signaling pathway. However, there are few studies on Csf3 in the PI3K-Akt signaling pathway, and the mechanism still needs further study. Fshr was another target gene of TCONS_00068680, which was associated with the p53 signaling pathway. Fshr was reported to be related to apoptosis and cell proliferation 50, but few studies investigated the mechanism of Fshr in the p53 signaling pathway in HIBD.
TCONS_00310736 was a lncRNA found to be up-regulated in the model group and down-regulated in the acupuncture group. Its target mRNAs, Th and Slc6a3, were related to dopaminergic synapses and Parkinson's disease. Dopaminergic synapses were found to be closely related to schizophrenia and Parkinson’s disease 51,52. The expressed protein of Th is the tyrosine hydroxylase, which was reported to inhibit the synthesis of dopamine and cause Parkinson’s symptoms by decreasing its enzymatic activity 53. Moreover, the inhibition of Th in the substantia nigra could decrease the movement frequency 54. Reportedly, Slc6a3 is strongly linked to dopamine transport. Hyperactivity, reduced sensitivity to reward, and impaired social behavior were measured in mutant rats with Slc6a3 knock-out 55. Htr1f was another target mRNA of TCONS_00310736 and was found to participate in the p53 signaling pathway. However, there are few studies on Htr1f in the p53 signaling pathway, and the mechanism still needs further study.
TCONS_00163999 with its target mRNA, Ccl12, associated with the TNF signaling pathway and NOD-like receptor signaling pathway, was also a lncRNA that was up-regulated in the model group and down-regulated in the acupuncture group. TNF plays an important role in nerve inflammation, and the TNF signaling pathway is closely related to ischemic stroke, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and multiple sclerosis 56. Reportedly, the functions of NOD-like receptors could be divided into four parts, namely, the inflammasome assembly, signaling transduction, transcription activation, and autophagy 57. Moreover, the NOD-like receptor protein 3 (NLRP3) inflammasome has been found to be closely associated with proptosis in encephalomyelitis 58. However, the mechanism of Ccl12 in the TNF signaling pathway and NOD-like receptor signaling pathway is not clear.
TCONS_00001104 was lncRNA down-regulated in the model group and up-regulated in the acupuncture group. Its target gene, Gykl1, is involved in the PPAR signaling pathway. Reportedly, activating the PPAR signaling pathway could significantly attenuate HI-induced brain injury by reducing neuronal apoptosis 59. However, there are a few studies on the function of Gykl1 in the PPAR signaling pathway. Il24, the other target gene of TCONS_00001104, was associated with the Jak-STAT signaling pathway. The expressed protein of IL-24 is interleukin-24, which was found to be closely related to apoptosis 60.
Additionally, we found that two lncRNAs (TCONS_00173912 and TCONS_00057072) up-regulated in the model group and two lncRNAs (TCONS_00088551 and TCONS_00097914) down-regulated in the acupuncture group jointly regulate a target gene, Reln. Interestingly, Reln was found to be up-regulated in the model group and down-regulated in the acupuncture group and was closely related to the PI3K-Akt signaling pathway. Reln is reported to play an important role in neuronal structure maintenance of mature neurons and can regulate neuronal migration and synaptogenesis 61,62. Moreover, some researchers found that Reln deficiency may be involved in the development of remote cognitive impairments and epigenetic regulation of DNA demethylation and histone acetylation of Reln might underlie the mechanisms of synaptic plasticity and memory retention in the medial PFC 63,64. Reln also has a role in PI3-kinase signaling in neuronal growth cones, and it contributes to final neuron positioning in the mammalian brain by local modulation of protein kinase B and glycogen synthase kinase 3beta kinase activities 65. The results of our study suggested that the increasing expression of TCONS_00173912 and TCONS_00057072 and the decreasing expression of TCONS_00088551 and TCONS_00097914 may inhibit Reln, which then works via the PI3K-Akt signaling pathway in neonatal HIBD.
In summary, we found several significant lncRNAs whose corresponding target genes might be the targets of acupuncture therapy for HIBD. At the same time, we investigated the miRNA–mRNA–lncRNA interaction network of the normal, HIBD, and acupuncture groups using RNA-seq analysis. To the best of our knowledge, this is the first research to study the expression of lncRNAs, circRNAs, and mRNAs between HIBD and acupuncture. The findings further expanded our understanding of ceRNA networks and will help us explore the functions of acupuncture. These novel networks may be potential biomarkers or therapeutic targets of acupuncture in HIBD. The research provides new perspectives on the mechanism of acupuncture and may affect the diagnosis and therapy of HIBD. However, this study also has its limitations because of its small sample size. In future studies, we will further expand the sample size and introduce clinical samples to provide a more sufficient basis for acupuncture treatment of HIBD.