| Objective:High-salt diet is not only a risk factor of hypertension, but also can cause target organ damage independent of blood pressure levels. Immune response involved in the incidence of atherosclerosis and hypertension process, of which acquired immune mechanisms are key factors in the inflammatory response-triggered target organ damage. Recent studies have shown that helper T cells17(T helper cell17, Th17)(generated interleukin-17) and regulatory T cells (Regulatory T cell, Treg)(expressing the transcription factor FoxP3) each play a pro-inflammatory and anti-inflammatory effects. Regulation of Th17/Treg immune balance is a hot research. Thl7/Treg immune imbalance caused inflammatory disorders, that is to say, destruction of the immune-inflammatory axis plays an important role in target organ damage. Recently, studies have indicated that high-salt diet can activate the immune system (such as Th17cells) to induce autoimmune disease, but whether the target organ damage is caused by Thl7/Treg immune imbalance has no relevant reports to date. Therefore, this study was designed to investigate the changes of Th17/Treg in response to short-term high salt intake in healthy volunteers, and their relationships with cardiovascular functional alteration.Methods:A total of15healthy male volunteers, and the average age of26.93±0.92yrs.(1) According to the World Health Organization (WHO), less than5g of salt a day (ie, NaC1) intake criteria is as a standard low-salt diet. With reference to our research group recently Tianjin residents fringe findings of average salt intake, standard high-salt diet is determined to be15g/day. Salt intake by the above criteria, the researchers provided all the volunteers the same food for eating in the same place. Entire dietary intervention time of17days were divided into three periods, including the baseline period (days0to3, volunteers eat in the same place according to personal taste freedom, a total of three days), immediately followed by high-salt period (days4to10, a total of7days), and finally into the low-salt period (days11to17, a total of7days). In addition to dietary intake of different amounts of salt, other nutrients, including protein and fat intake, are the same during the three periods, whereas to aviod movement, drinking, smoking, coffee and tea consume, and cannot add any extra food.(2) Systolic and diastolic blood pressure and heart rate were measured and recorded every day. At the end of the baseline, the first day of high salt, the7th day of high salt, the first day of low salt and the the7th day of low salt, urine and preipheral blood were collected for routine biochemical tests.(3) At day3of baseline, the first day of high salt, the7th day of high salt, the2th day of low salt and the the7th day of low salt, peripheral blood was harvested to evaluate Th17/Treg changes using a four-color (CD4, CD25, IL-17and FoxP3) flow cytometry platform. Using density gradient centrifugation, peripheral blood sample at the first day of high-salt period was collected for lymphocytes isolation, and then for gene expression profiling studies by gene chip technology, with results by principal component analysis, clestering analysis and gene set enrichment analysis. Total RNA of purified mononuclear cells was extracted using standard TRIzol procedure. PCR was used to analyze the mRNA expression of related pro-inflammatory and anti-inflammatory molecules.(4) A total of15participants finished serial magnetic resonanceimaging on day3,4,10,11and17using a Phillips Intera3.0Tesla whole body magnetic resonance system (Philips Medical Systems, Netherlands) with a six-channel flexible matrix coil to receive the MR signal. Coronal and axial T2*-weighted images were acquired inorder to covering two kidneys.(5) By non-invasive ultrasound of the brachial artery flow-mediated vasodilation (flow mediated dilation, FMD), we can accurately and quantitatively evaluate vascular endothelial function.Results:There was a good compliance of all the participants to dietary intervention, as determined by24-hour urine [Na+] during high-salt and low-salt period (all P<0.001).(1) Changes in the amount of urine, urine electrolytes and urine creatinine: compared with baseline,24hours urine amount, urine Na+, K+were significantly elevated at day7of high salt period (P all<0.001), and decreased significantly at 7th day of low-salt period (P<0.001). Compared with the7th day of high-salt, urine amount, urine Na+, K+reduced with statistically significant (P<0.001) at7th day of low-salt.24hours urine creatinine was significantly elevated (P<0.01) with high-salt, compared with that with baseline and low-salt.(2) Changes in blood pressure and heart rate:Compared with baseline, an upward trend in systolic and diastolic blood pressure at high-salt, and a downward trend at low salt, but the change was not statistically significant, while the heart rate of the high-and low-salt diet was significantly faster (P<0.05).(3) Compared with baseline (day3), the number of Th17cells measured on day4was decreased, which was accompanied by a reciprocal increase in Treg cell count, contributing to a decreased Thl7/Treg ratio (P<0.01). At the end of high-salt diet, there was a rebound in the number of Th17cells. In contrast, Treg cells presented a decreasing, which led to an increased Th17/Treg ratio (P<0.0\). The alteration in Th17/Treg ratio was regressed to baseline levels at the end of low-salt diet (day17).(4) The microarray analysis showed that compared with baseline level (day3), Thl7-related pathway was down-regulated at day4(P<0.01), while Treg-related pathway presented with an opposite change (P<0.01).(5) PCR results validate the microarray results:Inflammatory cytokines IL-23and IL-8expression decreased at day4(P<0.05); Th17pathway factor CD247downregulated (P<0.05); Inflammatory factors such as NFkB expression gradually increased with high salt load (P<0.05). NFAT5expression at day4increased at day4(P<0.05), with the comparable result of SGK-1(P<0.05).With the high salt load increase, inflammatory cytokines upregulated, and expression of Treg-associated inhibitory factor decreased.(6) Serial renal BOLD-MRI analyses showed a remarkable and progressive increase in renal medullary R2*signals (decreased tissue oxygenation) during high-salt loading (P<0.01). Notably, the increased renal medullary R2*signal regressed to its baseline level in a step-down fashion during low-salt feeding. At the end of low-salt phase, the cortical R2*signal was reduced compared with the high-salt period (P<0.01). (7) The FMD analysis showed that compared with the baseline and low-salt diet period, FMD was significantly higher at day4(P<0.05).Conclusions:Our study for the first time demonstrated a high salt diet can induce alterations in Thl7/Treg in humans, and these effects can be reversed by low-salt diet. This study suggested that short-term high salt intake can disturb the balance between Thl7-mediated pro-inflammatory and Treg-mediated anti-inflammatory response, which provides new evidence for the participation of immunity in high-salt intake related end organ injury. |
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