Effect Of Salt Taste Change On Salt Intake And Blood Pressure And Its Mechanism

Background and Aim:Excessive salt intake is an important risk factor for cardiovascular and cerebrovascular diseases such as hypertension,myocardial hypertrophy,atherosclerosis and stroke.The daily salt intake of Chinese population is much higher than the standard recommended by WHO.Salt intake reduction can significantly decrease the incidence and mortality of cardiovascular events,which is of great significance for the prevention and control of hypertension.Our country has made a lot of efforts to reduce salt intake,but in the past decades,the high intake of salt in people's daily diet has not changed significantly.Therefore,it is necessary to reveal the biological characteristics of organisms under long-term high salt intake and to explore new salt reduction measures.Taste is an important factor in determining our appetite and food intake.Taste sensitivity refers to the intensity of individual taste perception.People with higher taste sensitivity will perceive stronger taste under the same taste stimulus.Previous studies have suggested that changes in salt taste sensitivity are significantly correlated with salt intake.High salt intake could gradually reduce salt taste sensitivity,which might further maintain and increase high salt intake.Compared with normotensive people,the salt taste sensitivity in hypertensive ones is significantly reduced.Therefore,clarifying the mechanism of salt taste changes caused by long-term high salt intake is an urgent problem to be solved in exploring novel salt limitation strategies.The epithelial sodium channel?ENaC?has been proved to play an important role in salt taste perception.It is mainly expressed in taste cells of tongue fungiform papillae and responsible for salt taste perception of physiological range?low concentration,<150mM NaCl?.High concentration of salt solution?>200 mM?can activate bitter-and acid-sensing cells to induce aversion response thus maintaining a moderate salt intake.People who are extremely sensitive to bitterness are more sensitive to salt than those who are insensitive to bitterness.They also have a lower preference for high-salt foods and are more likely to eat low-sodium foods.Therefore,high salt diet may impair bitter taste perception,thereby reducing the high salt-induced aversive response,but the mechanism through which high salt affects bitter taste remains elusive.The perception of bitterness is produced by binding bitter molecules to G protein-coupled receptors located on the parietal membrane of type II taste receptor cells in taste buds.Transient receptor potential channel subfamily M member 5?TRPM5?is a voltage-dependent,selective Ca²⁺activated monovalent receptor potential channel specifically expressed in tongue epithelial cells.TRPM5 is the downstream molecule of bitter,sweet and umami taste information perception and transmission.These taste perception ability of TRPM5knockout mice was completely lost.It was found that high salt-activated bitterness-induced aversion was mediated by TRPM5,and knockout of TRPM5 could significantly inhibit the stimulation of high salt on bitter sensing cells.A series of previous studies conducted by our research group have shown that long-term high salt diet intervention affects the expression and function of various TRP channels,resulting in corresponding physiological disorders and target organ damage.However,whether long-term high salt consumption reduces the high salt-induced aversive taste perception,leading to the further increase of high salt intake by down-regulating the expression or function of TRPM5,still needs to be investigated.Chilli pepper is one of the most widely used flavoring agents in the world.The pungency of chilli pepper is a comprehensive sensation of heat and pain,which is produced by capsaicin,the main active ingredient in it.Animal studies have confirmed that dietary capsaicin improves endothelium-dependent vasodilation,antagonizes myocardial hypertrophy caused by high-salt diet,promotes urinary sodium excretion and plays an important role in the prevention and control of hypertension.Population epidemiological surveys also showed that people who prefer spicy food had a significantly lower risk of all-cause death than those who rarely eat spicy food.Other studies have shown that low concentrations of capsaicin can enhance salt taste perception without causing burning sensation.Therefore,it is worth examining whether spicy food consumption may reduce dietary salt intake and whether spicy flavor may affect salty-taste perception.This study consists of two parts.Firstly,we identified whether the molecular mechanism of high salt intake-induced hypertension was affecting the expression and function of TRPM5 through TRPM5 knockout(TRPM5^-/-)mice and wild-type?WT?mice,combined with the HEK-293 cell experiment transfected with TRPM5 plasmid.Secondly,we investigated the pungency preference through community cross-sectional study.The effects of pungency stimulation on salt taste preference,salt intake and blood pressure were also investigated in humans and animal models.This study will provide necessary theoretical support for the perfection and improvement of current salt restriction strategy.Materials and Methods:This study includes population survey,animal in vivo and in vitro experiments.In the first part,TRPM5^-/-mice and WT mice were used as the research objects.The effects of long-term high salt intake on salt-related aversive response were clarified by comparing the changes of salt taste,salt intake behavior and blood pressure before and after high salt diet intervention.The mechanism of high salt intake on the function of TRPM5 was preliminarily confirmed by cell experiments.In the second part,the salt intake,blood pressure and preference for salt taste and spicy diet were investigated through a cross-sectional study.Then,the effects of spicy stimulation on central salty taste perception were confirmed through a randomized,double-blind,interventional study.Finally,the effects of spicy stimulation on salt taste through the central nervous system and the potential mechanism were explored in animal models by optical fiber calcium signal fluorescence detection and optogenetic techniques.The experimental methods are as follows:1.The effects of high salt diet on preference for different concentration of salt solution and aversive reaction to high salt solution in mice were detected by licking response test and two-bottle taste preference test.2.The urine of mice was collected in metabolic cages,and the urine volume were recorded for the measurement of urinary sodium excretion.At the same time,the intake of food and water were recorded to determine the changes of sodium intake and excretion when salt taste changed.3.Non-invasive tail blood pressure and implant blood pressure telemetry technique were used to detect the changes of tail blood pressure and 24-hour ambulatory blood pressure in TRPM5^-/-mice and WT mice under high salt loading.4.Fluorescence ratio imaging system and patch clamp technique were used to detect TRPM5-mediated sodium uptake with intracellular sodium and calcium concentration in tongue epithelium of mice and cells under high salt preincubation.5.The effects of high salt diet on gene and protein expression levels of TRPM5 and PKC in mouse tongue epithelium and their interaction were determined by RT-PCR,western blot and co-immunoprecipitation.6.Subjects were recruited to determine their salt taste and spicy diet preferences,salt intake and blood pressure by means of taste tests and questionnaires.The metabolic changes of central gustatory cortex under different concentrations of salt solution and spicy stimulation were investigated by PET/CT imaging.7.Changes of neuronal activity in central taste-related brain areas of mice stimulated by different concentrations of salt solution and pungent stimulation were determined by optical fiber fluorescence calcium signal detection technique.8.By means of optogenetics,excitatory or inhibitory photosensitive proteins were expressed in the gustatory center of mice,and the changes of salt uptake behavior under excitation or inhibition were detected by licking response test.Results:1.Compared with mice fed with normal diet?ND?,mice fed with high salt diet?HSD?exhibited less aversion-avoidance reaction to high salt solution,weakened response in central bitter cortex to high salt stimulation,enhanced preference for salt-rich food,and significantly higher sodium intake and excretion in daily diet.2.Compared with ND mice,the gene and protein expression level and channel function of TRPM5 in tongue epithelium of HSD mice were significantly decreased,and the interaction between PKC and TRPM5 was weakened.In vitro experiments showed that the decrease of threonine phosphorylation level of PKC to TRPM5 in high salt circumstance was an important reason for the decline of its function.3.Compared with WT mice,TRPM5^-/-mice showed significantly reduced aversion-avoidance response to high salt solution,increased preference for high salt diet and higher blood pressure under high salt loading.The 24-hour dynamic systolic and diastolic blood pressure significantly increased after 12 weeks'high salt administration in TRPM5^-/-mice.4.The subjects with higher spicy preference had higher salt taste sensitivity,lower salt intake and lower blood pressure levels.Salt intake and salt taste preference were closely related to increased metabolic activity in insular and orbitofrontal cortex.Spicy stimulation could enhance the response to salt stimulation in the insular and orbitofrontal cortex,thus reversing the changes of salt taste intensity-dependent metabolic activity in these brain regions.5.The amplitude of calcium signal increased in a dose-dependent manner in the orbitofrontal cortex of mice stimulated by different concentrations of salt solution,which was affected by capsaicin administration.Excitation of the orbitofrontal cortex enhanced the preference for low salt and aversion to high salt,which was reversed after capsaicin was added to low salt solution.Conclusions:1.Long-term high salt intake down-regulates the expression and function of TRPM5in tongue epithelium,weakens the aversion-avoidance response to bitterness and salt stimulation,resulting in progressive increase of salt intake and hypertension.2.High salt affects the function of TRPM5 channel by lowers the activity of PKC and PKC-dependent threonine phosphorylation on TRPM5.3.Spicy diet enhances salt taste sensitivity,reduces salt intake and blood pressure.4.Salt intake and salt preference are closely related to the increase of metabolic activity in central insular and orbitofrontal cortex.Spicy stimulation enhances the metabolic activity of insular and orbitofrontal cortex under salt stimulation and modifies the salty taste information processed of in the brain.