SHRs were used in this manuscript as an animal model of essential hypertension to study the regulatory mechanism of reduced secretion of the SMG in hypertension. High-throughput sequencing of SMG lncRNAs and mRNAs from SHRs showed that there were 120 upregulated and 105 downregulated lncRNAs and 201 upregulated and 272 downregulated mRNAs compared with WKY rats.
First, GO and KEGG analyses of DE mRNAs were performed. KEGG analysis of the upregulated DE mRNA showed a higher degree of enrichment for inflammatory mediator regulation of TRP (transient receptor potential) channels (enrichment score: 3.583636). Transient receptor potential proteins have six transmembrane domains and act as ion channels with high Ca2+ permeability. TRP channels are composed of six subfamilies, including TRPC, TRPV, TRPM, TRPML, TRPP, and TRPA, in mammals25. An increasing number of studies have been carried out under the condition of various diseases, which highlights the role of Ca2+ signal transduction in the occurrence and development of diseases. Some of these Ca2+ entry channels are members of the TRP family. The secretion of fluid by the salivary glands can be stimulated by the activation of specific receptors on the cytoplasmic membrane of acini and mediated by the increase in cytosolic [Ca2+]26. Zhang et al.10 found that TRPV4 was a basal-lateral Ca2+ influx pathway in submandibular gland acinar cells, and its activation stimulated fluid secretion27. Radiation therapy and Sjogren’s syndrome both lead to salivary gland dysfunction and xerostomia. In both cases, abnormal Ca2+ signalling underlies the loss of fluid secretion28. Therefore, TRP channels may be related to salivary secretion. We found a significant decrease in salivary secretion in stimulated SHRs. KEGG analysis showed that 6 DE mRNAs participated in the inflammatory mediator regulation of TRP channels. This suggests that TRP channels may be involved in the process of decreased salivary secretion due to hypertension. It is worth noting that we found that TRP channels are subject to inflammatory mediator regulation. Hypertension is a chronic inflammatory process. The activation of the sympathetic nervous system and the renin–angiotensin–aldosterone system may activate the release of a large number of inflammatory factors and then damage vascular endothelial cells, leading to the rise of blood pressure29. Studies have reported that SHRs have a large number of activated lymphocytes and monocytes and release inflammatory factors. Based on our results, we speculated that the inflammatory process induced by hypertension affects the function of TRP channels, impairing Ca2+ signalling (aberrant Ca2+ signalling), which may be involved in the reduction of salivary secretion.
We identified 10 DE lncRNAs by qRT-PCR with the most significant expression differences in the SMG of SHRs, as follows: ENSRNOT00000057734, ENSRNOT00000075846, ENSRNOT00000093622, NR_144438, XR_341219, NR_131064, XR_339884, XR_590521, XR_591307, and XR_598156. We conducted in-depth analyses of these 10 lncRNAs, including CNC analysis and ceRNA analysis. Then, we performed GO and KEGG analyses according to predicted target genes to identify related molecules and pathways regulated by these DE lncRNAs. We observed an interesting phenomenon in which many of these predicted target genes were related to the immune response. The GO analysis of the CNC network revealed that the most related BP was the immune response: immune response-regulating cell surface receptor signalling pathway (enrichment score 7.97915179902155), immune response-regulating signalling pathway (enrichment score 6.75444880796142), positive regulation of the immune response (enrichment score 6.66100197807179), the regulation of cytokine production (enrichment score 6.1222877173628), and positive regulation of cytokine production (enrichment score 6.0192956725254). KEGG analysis of the CNC network showed that antigen processing and presentation were highly enriched (enrichment score 3.25377). This suggested that the 10 DE lncRNAs identified by us were involved in the regulation of immune processes. However, the pathway by which lncRNAs regulate these mRNAs needs further study.
We also carried out ceRNA analysis to establish the relationship between these 10 DE lncRNAs and DE mRNAs. GO analysis of the downstream target genes predicted by ceRNA revealed that the top BPs involved were as follows: the regulation of cytokine production involved in the immune response (3.79663529907157) and positive regulation of cytokine production involved in the immune response (3.64351452795962). KEGG analysis also showed that antigen processing and presentation were significantly enriched (enrichment. Score 2.306039). This further suggested that lncRNAs regulated these BPs through miRNAs. This provides basic data for further research in the future.
The immune system may have been a key factor in the development of hypertension. The general assumption is that the accumulation of immune cells in blood vessels (especially perivascular fat) and the kidneys, heart and brain will promote a chronic inflammatory response, thus damaging the blood pressure regulation function of these organs, leading to hypertension30. It has been reported that circulating IgG levels in patients with hypertension are higher than those in individuals with normal blood pressure31,32. The mechanism of antibodies promoting hypertension occurs through the direct activation of receptors and channels regulating vascular tension, renal sodium reabsorption and cardiac function33. Cytotoxic T lymphocytes may also affect blood pressure by enhancing renal reabsorption of sodium and water34,35. T-helper cells may directly affect the activity of cyclooxygenase in the vascular wall, thus promoting endothelial dysfunction and increasing vascular resistance36. Abnormal immune activation plays a pathogenic role in the development of animal and human hypertension. SHRs had many autoantibodies in addition to decreased salivation. High levels of autoantibodies against the second extracellular loop of the a1-adrenoceptor (a1-AR autoantibody, a1-AA) are found in SHRs, and this altered responsiveness is due to endothelial dysfunction and decreased NO bioavailability37. Chen CH et al. observed an elevation of the plasma concentration of IgA and of circulating IgA autoantibodies to single-stranded DNA, double-stranded DNA, and thyroglobulin in SHRs38. At a prehypertensive age (1 month), anticardiolipin antibody levels in SHRs were significantly higher than those in control Wistar rats39. These suggested that an abnormal immune activation occurred in SHR. Combined with our experimental results, we believed that immunotargeted therapy will become a new target and new idea to reduce the complications of hypertension, including the reduction of salivation.
The secretory function of the salivary gland is closely related to the function of the immune system. Primary Sjogren’s syndrome (PSS), systemic lupus erythematosus, rheumatoid arthritis and other systemic autoimmune diseases are usually accompanied by decreased saliva secretion40. PSS is characterized by chronic inflammation of the salivary and lacrimal glands, respectively, followed by dry oral and ocular mucosa41. The epithelial cells of the exocrine organs of PSS patients showed a large amount of lymphocyte infiltration. A T cell-related proinflammatory microenvironment and subsequent chronic inflammation lead to irreparable structural modifications and the hypofunction of the target organs, including insufficient secretion of salivary glands and severe dry mouth. At the same time, this process is also regulated by ncRNAs. The upregulated lncRNA-PVT1 in CD4+ T cells of PSS patients can maintain the expression of Myc to control the proliferation and effector functions of CD4+ T cells by regulating the reprogramming of glycolysis42. Decreased saliva secretion can affect oral health. Clinical studies report that high blood pressure reduces saliva secretion in patients43,44,45. Saliva plays a key role in digestion, taste, cleaning, the hydration of the oral mucosa and tooth protection and is essential to maintain the dynamic balance of the oral environment. When the secretory function of the salivary gland is damaged, the decrease in salivary secretion will affect the oral microenvironment, leading to dry mouth, taste disorders, rickets, periodontal disease, and chewing and swallowing difficulties, which decrease the quality of life. In addition, insufficient saliva secretion will lead to a decrease in the oral clearance rate, a decrease in the saliva pH value and buffering ability, and a decrease in the immune defence ability. These symptoms may increase the risks of oral diseases such as cervical caries, periodontitis and oral candidiasis46,47. Considering the importance of oral health, we should pay ample attention to the decrease in saliva secretion caused by hypertension. This study shows that many mRNAs in the SMG of SHRs are related to the immune response, and these molecules are regulated by lncRNAs. There are obviously aberrant immune reactions that are regulated by lncRNAs in the SMG in hypertension, and these aberrant reactions provide a new idea for the treatment of hypertension complications. The 10 lncRNAs that we identified are worthy of further study.
RNA sequencing is a powerful tool that can not only decipher non-annotated transcriptional activity but also reveal the differential expression profile of RNAs underlying specific phenotypic differences. In this study, we screened the genome-wide expression profiles of lncRNAs and mRNAs in the SMG in SHRs and found that the expression of lncRNAs and mRNAs in SMG tissues was significantly different from that of the control group, and some abnormal expression of lncRNAs may play important roles in the development and progression of hyposalivation. We show for the first time that TRP mRNAs were significantly upregulated in the SMG of SHRs, which may serve as a novel therapeutic target. We also found that lncRNAs regulate immune responses, providing a new direction to control immune responses. A potential drug that regulates one key lncRNA may be useful to suppress the occurrence of hyposalivation in hypertension. In addition, some immunosuppressors can be used to improve hyposalivation. Our study had some limitations. First, the sample size of the microarray analysis used to verify the results was small. Second, the subsequent functional verification needs to be further improved.
In the past 50 years, researchers have concentrated on the role of lncRNAs in disease processes. Disease–lncRNA association inference is important in the design of specific molecular tools for human disease diagnosis, treatment, prognosis and prevention. It is difficult to quickly and efficiently study the relationship between lncRNAs and diseases by relying only on traditional biological experiments. Chen et al. developed the powerful computational model of LRLSLDA to predict potential disease-related lncRNAs based on a semi-supervised learning framework48. To date, dozens of computational models have been proposed for this purpose. In general, the current computational models for lncRNA function prediction could be classified into four categories: sequence alignment-based models, gene co-expression-based models, lncRNA–miRNA/mRNA/protein interaction-based models and integrative feature-based models. Computational models could be effective ways to identify potential lncRNA functions and lncRNA–disease associations, hence decreasing the time and cost of biological experiments. In our study, high-throughput sequencing was performed in the SMG, followed by bioinformatics analysis using a computer model. This can help us quickly and efficiently identify lncRNAs associated with hyposalivation in hypertension.
In summary, we constructed the expression profile of DE lncRNAs and DE mRNAs in the SMG of SHRs. mRNA profiling revealed that inflammatory mediator regulation of TRP mRNAs was significantly upregulated in the SMG of SHRs. Our CNC and ceRNA network analyses suggested that aberrant immune responses were observed in hypertensive SMGs and were regulated by 10 DE lncRNAs. Our findings further expand our understanding of the role of lncRNAs in salivary secretion. Through the construction of the regulatory networks, our study is helpful to understand their role in the pathological process of hyposalivation in hypertension. This study may provide valuable clues for further research, as the role of lncRNAs has not been fully uncovered in hyposalivation in hypertension.
