MARGARIC ACID REDUCES PAIN MEDIATED BY PIEZ02

Abstract

This description describes a method for treating pain by administering a pharmaceutical composition containing a therapeutically effective amount of margaric acid to a subject requiring pain treatment. Pharmaceutical compositions, such as topical and transdermal compositions, containing margaric acid and a pharmaceutically acceptable excipient are also described. A pain-relieving composition containing margaric acid, eicosapentaenoic acid, and a pharmaceutically acceptable excipient is further described.

Description

MARGARIC ACID REDUCES PIEZ02-MEDIATED PAIN CROSS-REFERENCE WITH RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 976,014, filed February 13, 2020, which is incorporated by reference in its entirety herein. DESCRIPTION FIELD The present disclosure relates to methods of pain treatment using dietary fatty acids. BACKGROUND The skin is innervated by sensory neurons that express mechanosensitive ion channels, enabling the detection and discrimination of pleasurable from painful touch. The mechanosensitive ion channel PIEZO2 is highly expressed in sensory neurons and Merkel cells, where it mediates light touch (i.e., rubbing) and vibration. Importantly, research has also shown that PIEZO2 contributes to tactile allodynia (i.e., when innocuous sensations become painful under inflammatory conditions). Mechanosensitive ion channels are known to be modulated by the mechanical properties of the membrane, intracellular and extracellular proteins, and / or cytoskeletal elements. Several lines of evidence suggest that PIEZO2 channels interact with cellular components to fulfill their physiological function. For example, the association of PIEZO2 with stomatin-like protein 3 and cholesterol increases its sensitivity to mechanical stimuli, sensitization by inflammatory agents such as bradykinin, and regulation by phosphoinositide lipids. Interestingly, PIEZO2 requires cytoskeletal elements such as actin and tubulin for normal function. Together, these data indicate complex interactions that work together to fine-tune PIEZO2 function. Recent findings that Piezol-deficient humans and gene knockout mice do not develop sensitization or painful reactions to innocuous touch after skin inflammation suggest that targeting this receptor may be a viable strategy to treat tactile allodynia. What is needed are chemical compounds that interact with Piezo receptors and use i? LRRnn / zznz / E / YiAi to mediate Piezo receptor function. BRIEF SUMMARY In one aspect, the method for treating pain comprises administering to a subject in need of pain treatment a pharmaceutical composition comprising a therapeutically effective amount of margaric acid. In another aspect, a pharmaceutical composition comprises margaric acid and a pharmaceutically acceptable excipient. In yet another aspect, a composition for the treatment of pain comprises margaric acid, eicosapentaenoic acid and a pharmaceutically acceptable excipient. BRIEF DESCRIPTION OF THE FIGURES Figures 1a-1f show that margaric acid (MA) inhibits mouse PIEZO2 currents expressed heterologously in N2A7''ezo7·4 cells. Figure 1a shows representative recordings of whole-cell membrane clamping evoked by mechanical stimulation (at -60 mV) of control and Piezo2 variant 2 (V2)-transfected ^A^07'4 cells treated with margaric acid (MA) (1, 50, 200, 300, 400, and 600 μM). Figure 1b shows the normalized current densities evoked by the maximal displacement of Piezo2 V2-transfected N2A7>'Z44 cells treated with MA. A Boltzmann function, Eq. (2), was fitted to the data (IC50 =190.6 ± 14.7 SEM). Circles are mean + SD. n is denoted on the x -axis in panel c. The box plots in Figure 1c show the mean, median, and 75th to 25th percentiles of the displacement thresholds required to elicit currents in the P1EZO2 V2 control and ^A737'4 cells.n is denoted on the x-axis. One-way ANOVA and Bonferroni test. Figure Id shows representative PIEZO2 currents (at -60 mV) of control and Piezo2 V2-transfected N2A7í'z4a cells treated with MA (50 μM every day for 4 days). Figure le shows the peak displacement-evoked PIEZO2 V2 current densities of control and ^A7^'7-4 cells treated with MA (50 μM for 18 h, and every day for 4 days), n is denoted on the x-axis. One-way ANOVA and Bonferroni test. The boxplots in Figure 11 show the mean, median, and 75th to 25th percentiles of the displacement thresholds required to elicit currents from control PIEZO2 V2 and PIEZO2 V2-transfected Ν2Α7>'ΖΛ4 cells treated with MA (50 μM each day for 4 days), n is denoted on the x-axis. Paired t-test. Asterisks indicate values ​​significantly different from control (***p<0.001) and nsindicates values ​​that are not significantly different from the control. Figures 2a-2g support that MA inhibits mouse PIEZO2 currents heterologously expressed in ^A7'277 cells. Figure 2a shows the peak displacement-evoked current densities of control and MA-treated (1, 25, 50, 100, 200, 300, 400, and 600 μM) N2A / '?Z / / ' cells transfected with the Piezo2 variant (V2). Bars are mean + SD. n is denoted on the x -axis. Unpaired t -test, unpaired t -test with Welch's correction, and Mann-Whitney test. Figure 2b shows the PIEZO2 V2 inactivation time constants caused by the peak displacement of control and MA-treated cells (1, 25, 50, 100, 200, 300, 400, and 600 μM). Bars are mean + SD. N is denoted on the x -axis. One-way ANOVA and Bonferroni test.Figure 2c shows the PIEZO2 V2 inactivation time constants evoked by maximal displacement of control and N2APiezol- / - cells treated with MA (50 μM each day for 4 days). Bars are mean + SD. n is denoted on the x-axis. Unpaired t-test. Figure 2d shows representative currents (at -60 mV) of control and Piezo2 N2-transfected ^A772077 cells treated with MA (25 μM each day for 8 days). Figure 2e shows the PIEZO2 V2 current densities evoked by maximal displacement of control and N2AL£2ο7 α cells treated with MA (25 μM each day for 8 days), n is denoted on the x-axis, unpaired t-test.The box plots in Figure 2f show the mean, median, and 75th to 25th percentiles of the displacement thresholds required to elicit control PIEZO2 V2 and Piezo2 N2-transfected ^A^207 7 cells treated with MA (25 μM each day for 8 days), n is denoted on the x -axis. Unpaired t -test. Figure 2g shows the inactivation time constant of PIEZO2 V2 elicited by the maximal displacement of control and N2A7'Í'2°7'7 cells treated with MA (25 μM each day for 8 days). Bars are mean + SD. n is denoted on the x -axis. Unpaired t -test. Asterisks indicate values ​​significantly different from the control (***p<0.001 and **p<0.01) and ns indicates not significantly different from the control. Figures 3a-3i support that MA inhibits mouse P1EZO2 currents heterologously expressed in N2A7íC2°77 cells. Figure 3a is a schematic representation of missing (black) and / or present (white) exons of Piezo2 variants (V). Figure 3b shows representative recordings of whole-cell membrane attachment from control and Piezo2 V14-transfected N2APiezol cells treated with MA (300 μM; 18 h). Figure 3c i? LRRnn / zznz / E / YiAi shows the peak displacement-evoked current densities of control and Piezo2 V14-transfected cells treated with MA (300 μM; 18 h). Bars are mean + SD. n is denoted on the x -axis. Mann-Whitney test.The box plots in Figure 3d show the mean, median, and 75th to 25th percentiles of the displacement thresholds required to elicit currents from control and Piezo2 V14-transfected N2Ap'2o77' cells treated with MA (300 μM; 18 h). n is denoted on the x -axis. Unpaired t -test. Figure 3e shows the maximal displacement-elicited inactivation time constants from control and Piezo2 V14-transfected N2Ap'2o77' cells treated with MA (300 μM; 18 h). Bars are mean ± SD. n is denoted as M. Figure 3f shows representative whole-cell membrane clamping recordings of control and Piezo2 VI6-transfected ^A^77 cells treated with MA (300 μM; 18 h). Figure 3g shows the peak displacement-evoked current densities of control and Piezo2 V16-transfected ΝIΑ^01'1'' cells treated with MA (300 μM; 18 h). Bars are mean ± SD.n is denoted on the x-axis. Unpaired t-test with Welch's correction. Box plots in Figure 3h show the mean, median, and 75th to 25th percentiles of the displacement thresholds required to elicit currents in control and Piezo2 V16-transfected N2A / ''<'Z77' cells treated with MA (300 μM; 18 h). n is denoted on the x-axis. Mann-Whitney test. Figure 3i shows the maximal displacement-elicited inactivation time constants in control and Piezo2 VI-transfected ^A^2077' cells treated with MA (300 μM; 18 h). Bars are mean + SD. n is denoted on the x-axis. Unpaired t-test. Asterisks indicate values ​​significantly different from the control (**p<0.01 and *p<0.05) and ns indicates not significantly different from the control. Figures 4a–4f show that latrunculin A enhances P1EZO2 inhibition by MA. Figure 4a shows the normalized current density evoked by the maximal displacement of N2A cells supplemented with MA (1, 10, 25, 50, 100, and 300 μM; 18 h) (expressing endogenous Piezol) and Piezo2-transfected N2A / Vz / 7' cells supplemented with MA (1, 25, 50, 100, 200, 300, 400, and 600 pM; 18 h) (triangles and circles, respectively). A Boltzmann function, Eq. (2), is fitted to the data (P1EZO1 IC50 = 28.3 + 3.48 SEM; PIEZO2 IC50 = 190.6 ± 14.7 SEM). Symbols are mean + SD. Figure 4b shows the normalized current densities evoked by the maximal displacement of N2APiezo17' cells transfected with PIEZO2, which is supplemented with MA (1, 25, 50, 100, 200, 300, 400 and 600 μM; 18 h) that are treated with and without latrunculin A. A Boltzmann function, Eq. (2), is fitted to the data (MA IC50 = L ΑΑΩΠ / 77Π7 / Ε / ΥΙΛΙ 190.6 ± 14.7 SEM; MA + LatA IC50 = 75.4 ± 13.3 SEM). Circles are mean + SD. Figure 4c shows the normalized current densities evoked by the maximal displacement of N2A cells supplemented with MA (1, 10, 25, 50, 100, and 300 μM; 18 h) (expressing endogenous Piezol). A Boltzmann function, Eq. (2), is fit to the data (MA IC50 = 28.3 + 3.4 SEM; MA + LatA IC50 = 25.6 + 8.4 SEM). Triangles are mean + SD. Figure 4d shows a ribbon representation of the P1EZO2 monomer (PDB ID: 6KG7; grey) highlighting the residues that are exchanged for those of PIEZOL. Figure 4e shows the inhibition of N2A cells and Ν2ΑΛί>ζ'?ΛΛ cells transfected with Piezo2 and Piezo2-Piezol bundle chimera, which are supplemented with MA (100 μM), n is denoted on the x-axis. Unpaired t-test and Mann-Whitney test.Figure 4f shows normalized current densities evoked by the maximal displacement of N2A cells (expressing endogenous Piezol) and N2Apzo / / cells transfected with Piezo2 and Piezo2-Piezol beam chimera supplemented with MA (100 pM; 18 h) and treated with and without latrunculin. n is denoted on the x -axis. Unpaired t -test (for PIEZO1) and Mann-Whitney test (for PIEZO2 and PIEZO2-Piezol beam chimera). Asterisks indicate values ​​significantly different from the control (**p<0.01 and ***p<0.001) and ns indicates values ​​that are not significantly different from the control. Figures 5a-5d also show that latrunculin A enhances PIEZO2 inhibition by MA. Figure 5a shows representative recordings of whole-cell membrane clamping evoked by mechanical stimulation (at -60 mV) of control and Piezo2 V2-transfected N2Ap'z / _ / · cells supplemented with MA (1, 25, 50, 100, 200, 300, 400, and 600 pM) treated with and without Latrunculin A, Figure 5b shows the peak displacement-evoked current densities of control and Piezo2 N2-transfected N2Ap'z / _ / · cells supplemented with MA (1, 25, 50, 100, 200, 300, 400, and 600 pM) treated with and without Latrunculin A. n is denoted on the x -axis. Unpaired t-test, Mann-Whitney test, and unpaired t-test with Welch correction.Figure 5c shows representative whole-cell membrane clamping recordings evoked by mechanical stimulation (at -60 mV) of control and N2A cells supplemented with MA (1, 10, 25, 50, 100, and 300 pM) (expressing endogenous Piezol), treated with and without latrunculin A. Figure 5d shows the peak displacement-evoked current densities of control and N2A cells supplemented with MA (1, 10, 25, 50, 100, and 300 pM) (expressing endogenous Piezol) treated with and without latrunculin A. n is denoted on the x -axis. Unpaired t -test with Welch’s correction, unpaired t -test, and Mann-Whitney test. Asterisks indicate 5 i? LRRnn / zznz / E / YiAi values ​​significantly different from the control (***p<0.001, **p<0.01 and *p<0.05) and ns indicates not significantly different from the control. Figures 6a-6e also show that latrunculin A enhances PIEZO2 inhibition by MA. Figure 6a shows representative whole-cell membrane pinning recordings evoked by mechanical stimulation (at -60 mV) from control, Piezo2-Piezol beam chimera-transfected N2AAt'zo / / ' cells treated with MA (100 μM for 18 h) with and without latrunculin A (1 μM for 1 h). Figure 6b shows the current-voltage relationship of the mechano-dependent currents of the P1EZO2-P1EZO1 chimera as determined by whole-cell membrane pinning experiments. Circles are mean + SD. n = 6. The box plots in Figure 6c show the mean, median, and 75th to 25th percentiles of the displacement thresholds required to evoke currents from control, Piezo2-Piezol beam chimera-transfected Ν2Αρ'£ζ',Λ / · cells treated with MA (100 μM for 18 h), with and without Latrunculin A (1 μM for 1 h).n is denoted on the x-axis. Mann-Whitney test (for control vs. MA and control vs. MA + LatA) and unpaired t-test (for MA vs. MA + LatA). Figure 6d shows the maximal displacement-evoked inactivation time constants of Piezo2 V2 and Piezo2-Piezol beam chimera transfected in N2Ap'<,zr / \ cells. Bars are mean ± SD. n is denoted on the x-axis. Mann-Whitney test. Figure 6e shows the maximal displacement-evoked current densities of control, Piezo2Piezol beam chimera-transfected N2A / νζΛΛ cells treated with MA (100 μM for 18 h) with and without Latrunculin A (1 μM for 1 h). Bars are mean ± SD. n is denoted on the x-axis. Mann-Whitney test (for control vs. MA + LatA and MA vs. MA + LatA) and unpaired t test (for control vs. MA). Asterisks indicate values ​​significantly different from the control (**p<0.01 and ***p<0.001) and nsindicates that it is not significantly different from the control. Figures 7a–7f show that MA decreases mechanically activated currents in mouse MCC13 and DRG neurons. Figure 7a shows representative mechanically stimulated (-60 mV) whole-cell membrane-clamp recordings of fast (x < 10 ms), intermediate (10 < t < 30 ms), and slow (t > 30 ms) inactivating currents from control (top) and MA-treated MCC13 (300 pM) (bottom). Figure 7b shows peak displacement-evoked current densities of control and MA-treated MCC13 cells (300 pM). Bars are mean + SD. n is denoted on the x -axis. Unpaired t -test. Box plots in Figure 7c show the mean, median, and 75th–25th percentiles of 6 i? LRAnn / zznz / E / YiAi the displacement thresholds required to elicit mechanical currents of control and MCC13 cells treated with MA (300 pM), n is denoted on the x-axis.Unpaired t test with Welch's correction. Figure 7d shows representative whole-cell membrane-clamp recordings evoked by mechanical stimulation (at -60 mV) of fast (x < 10 ms), intermediate (10 < t < 30 ms), and slow (x > 30 ms) inactivating currents from control (top) and DRG neurons treated with MA (300 μM) (bottom). Figure 7e shows peak displacement-evoked current densities of control and DRG neurons treated with MA (300 μM). Bars are mean ± SD. n is denoted on the x axis, unpaired t test. The box plots in Figure 7f show the mean, median, and 75th to 25th percentiles of the displacement thresholds required to evoke mechanical currents in control and MA-treated DRG neurons (300 pM), n is denoted on the x -axis. Unpaired t -test with Welch's correction. Asterisks indicate values ​​significantly different from control (***p<0.001, **p<0.01 and *p<0.05) and ns indicates values ​​that are not significantly different from the control. Figures 8a-8d show that MA decreases mechanically evoked action potentials in mouse DRG neurons. Figure 8a shows representative current-clamp recordings of mechanically evoked membrane potential changes in control and MA-treated (300 μM) DRG neurons (up to 10 and 15 μM indentation, respectively). Figure 8b shows the peak membrane potential versus mechanical indentation in independent control (n = 10) and MA-treated (n = 7) DRG neurons. The top panel shows box plots showing the mean, median, and 75th to 25th percentiles of the threshold displacement required to evoke an action potential in these neurons. Unpaired t test. Figure 8c shows representative current-clamp recordings of membrane potential changes evoked by a 1 Hz mechanical stimulus control train and MA-treated DRG neurons.The inset illustrates the progressive decrease in membrane potential with increasing pulse number. Figure 8d shows on the left: the number of action potentials evoked per sweep versus mechanical indentation in control (n = 6) and MA-treated DRG neurons (n ​​= 6). Right: rectangular diagram showing the action potentials evoked by 12 pm of indentation. Bars indicate stimuli that evoked action potentials. Columns and rows represent cells and sweeps, respectively. Asterisks indicate values ​​significantly different from the control (***p<0.001). L ΑΑΩΠ / 77Π7 / Ε / ΥΙΛΙ Figures 9a–9g show that MA does not alter the electrical excitability of mouse DRG neurons. Figure 9a shows representative whole-cell membrane-clamp recordings from control and DRG neurons treated with MA (300 pM) depolarized in a step-like manner from a holding potential of −80 mV. Figure 9b shows the normalized inward current densities evoked by step depolarization from a holding potential of DRG neurons treated with −80 mV of control (n = 7) and MA (n = 7; 300 μM). Circles are mean + SD. Figure 9c shows the normalized outward current densities evoked by step depolarization from a holding potential of DRG neurons treated with −80 mV of control (n = 7) and MA (n = 7; 300 μM). Circles are mean + SD.Figure 9d shows the membrane potential values ​​recorded just after whole-cell configuration is achieved from control and DRG neurons treated with MA (300 pM), n is denoted on the x-axis, Mann-Whitney test. Figure 9e shows the representative current-clamp recording of membrane potential changes evoked by current injection in control and DRG neurons treated with MA (300 pM). Figure 9f shows the action potential amplitudes evoked by current injection from control and DRG neurons treated with MA (300 pM), and n is denoted on the x-axis. Unpaired t-test with Welch's correction. The box plot in Figure 9g shows the mean, median, and 75th to 25th percentiles of the minimum injected current that elicits action potentials from control and DRG neurons treated with MA (300 pM), n is denoted on the x axis.Mann-Whitney test. ns indicates values ​​not significantly different from the control. Figures 10a–10d show that MA recovers normal mechanical responding in sensitized mouse DRG neurons. Figure 10a shows representative whole-cell membrane-clamp traces of mechanically activated currents after consecutive perfusion of bath solution (60 s) and bath solution containing bradykinin (BK; 1 pM) into control and MA-treated DRG neurons (300 pM; 18 h). Figure 10b shows the current densities evoked by 10 pM displacement of control and MA-treated DRG neurons (300 pM; 18 h) perfused with bath solution (60 s) and with bath solution containing bradykinin (BK; 300 s, 1 pM) consecutively. Bars are mean ± SD, data samples are paired, n is indicated on the x -axis. Paired t-test, unpaired t-test, Mann-Whitney and Wilcoxon matched-pairs signed-rank test.Figure 10c shows representative whole-cell membrane-clamp recordings of 8 i? LRRnn / zznz / E / YiAi evoked by mechanical stimulation (at -60 mV) fast (t < 10 ms), intermediate (t < 30 ms), and slow (τ > 30 ms) inactivating currents from control and DRG neurons treated with BK (1 μM for 1 Bit) and BK+MA (1 μM and 300 μM, respectively, for 18 h). Figure 10d shows the peak displacement-evoked current densities of fast (τ < 10 ms), intermediate (τ < 30 ms), and slow (τ > 30 ms) inactivating currents evoked by mechanical stimulation (at -60 mV) of control and DRG neurons treated with BK (1 μM; 18 h) and BK+MA (1 μM and 300 μM, respectively; both 18 h). Bars are mean + SD. Unpaired t test, n is denoted on the x axis. Asterisks indicate values ​​significantly different from control (***p<0.001, **p<0.01, and *p<0.05) and ns.indicates that it is not significantly different from the control. Figure 11 also shows that MA restores normal mechanical response in sensitized mouse DRG neurons. Figure 11 shows the current and fold change of control and MA-treated DRG neurons (300 μM; 18 h) perfused for 60 s with bath solution and 300 s with bath solution containing bradykinin (BK; 1 μM) consecutively, n is denoted below the bars. Unpaired t test, ns indicates not significantly different from control. Figures 12a-12d show that MA administration protects against tactile allodynia in mice. Figure 12a is a cartoon depicting the repeated injury paradigm. Mice were tested for their baseline withdrawal thresholds to mechanical stimulation (von Frey) and radiant heat (Hargreaves). Subsequently, one hindpaw was injected daily for 1 week with a small volume of saline or saline containing MA (5 mM). After which, mechanical and thermal withdrawal thresholds were measured again with or without the acute inflammation evoked by the application of mustard oil (AITC). Figure 12b shows that repeated injection of saline for 7 days caused hyperreactivity to mechanical stimuli.Under baseline conditions (open circles), mice begin to withdraw their paws in most trials (out of 10) when the von Frey filament force reaches between 0.2-0.4 g. In contrast, after repeated lesions (closed circles), mice withdraw much more frequently even to very light filaments (0.02 g). Significant hyperresponsiveness is observed for all filaments > 0.02 g until maximal responses are evoked (10 / 10 responses at 0.6-1 g). These allodynic-like responses do not develop when mice are treated with MA (compare open and closed circles). **p>0.01; n=6 mice per treatment group (n=9 i? LRRnn / zznz / E / YiAi males and 3 females). Figure 12c shows repeated saline injection decreases the response latency(ies) to radiant heat focused on the injured paw as well as on the contralateral side.The inclusion of MA in the injection solution had no effect on this heat hypersensitive response, n = 6 mice per treatment group (n = 3 males and 3 females). Figure 12d shows that acute topical administration of mustard oil (AITC) did not increase the hypersensitive response to punctate stimuli after 1 week of MA treatment (compare circles with squares). As a control, mice injected with saline than treated with AITC responded significantly more frequently to low-strength filaments (squares) n = 6 mice per treatment group (n = 3 males and 3 females). Figures 13a-13h show that MA decreases mechanically activated currents in human iPSC-derived neurons. Figure 13a is a micrograph showing a human iPSC-derived neuron in whole-cell membrane clamp ready for mechanical stimulation. Figure 13b shows representative whole-cell membrane clamp traces of mechanically activated currents from control and iPSC-derived neurons treated with MA (300 and 600 μM for 18 h and 50 μM each day for 5 days). Figure 13c shows the peak displacement-evoked current densities of control and iPSC-derived neurons treated with MA (300 and 600 μM for 18 h and 50 μM each day for 5 days). Bars are mean ± SD. n is denoted on the x -axis. Unpaired t-test.The box plots in Figure 13d show the mean, median, and 75th–25th percentiles of the displacement thresholds required to evoke currents in control and iPSC-derived neurons treated with MA (300 and 600 pM for 18 h and 50 pM each day for 5 days), n is denoted on the x -axis. Unpaired t -test. Figure 13e shows the time constant of PIEZO2 inactivation evoked by maximal displacement in control and iPSC-derived neurons treated with MA (300 and 600 pM for 18 h and 50 pM each day for 5 days). Bars are mean ± SD. n is denoted on the x -axis. Unpaired t -test. Figure 13f shows representative whole-cell membrane clamp recordings from control and depolarized iPSC-derived neurons treated with MA (300 pM) in a stepwise manner starting from a membrane potential of -80 mV.Figure 13g shows the normalized inward current densities evoked by -80 mV step depolarization of control (n = 8) and DRG neurons treated with MA (n = 8; 300 μM). Circles are mean ± SD. Figure 13h shows the normalized outward current densities evoked by 80 mV step depolarization of control (n = 8) and DRG neurons treated with MA (n = 8; 300 μM). Circles are mean ± SD. Asterisks indicate values ​​significantly different from control (***p<0.001 and **p<0.01) and ns indicates values ​​that are not significantly different from control. Figures 14a-14d also show that MA decreases mechanically activated currents in neurons derived from human iPSCs.Figure 14a shows representative whole-cell membrane-clamp recordings evoked by mechanical stimulation (at -60 mV) from control and human Piezo2-transfected ^A^2'777 cells supplemented with MA (300 pM). Figure 14b shows the maximal displacement-evoked current densities from control and human Piezo2-transfected ^A^077' cells treated with MA (300 pM; 18 h). Bars are mean + SD. n is denoted on the x -axis. Unpaired t -test. The box plots in Figure 14c show the mean, median, and 75th to 25th percentiles of the displacement thresholds required to evoke currents from control and human Piezo2-transfected ^A^077' cells treated with MA (300 pM; 18 h). n is denoted on the x-axis. Unpaired t-test.Figure 14d shows the peak shift-induced inactivation time constants of control and human Piezo2-transfected Ν2ΑΡί<ζΛ / · cells treated with MA (300 pM; 18 h). Bars are mean + SD. n is denoted on the x -axis. Unpaired t -test. Asterisks indicate values ​​significantly different from the control (***p<0.001) and ns indicates not significantly different from the control. Figures 15a-15g further show that MA decreases mechanically activated currents in rat DRG neurons. Figure 15a is a micrograph showing a rat DRG neuron in the whole-cell patch-clamp configuration ready for mechanical stimulation. Figure 15b shows representative whole-cell membrane-clamp traces of mechanically activated currents from control and rat DRG neurons treated with MA (300 pM for 18 h). Figure 15c shows the maximal displacement-evoked current densities of control and rat DRG neurons treated with MA (300 for 18 h). Bars are means ± SD. n is denoted on the x -axis. Unpaired t -test.The box plots in Figure 15d show the mean, median, and 75th–25th percentiles of the shift thresholds required to evoke currents in control and MA-treated rat DRG neurons (300 pM for 18 h), n is denoted on the x -axis. Unpaired t -test. Figure 15e shows representative current-clamp recordings of the membrane potential changes evoked by mechanical stimulation in control and MA-treated rat DRG neurons (300 pM) and (up to 9 and 13 pM indentation, respectively). Figure 15f shows the peak membrane potential against mechanical indentation of independent control (η = 11) and MA-treated DRG neurons (n ​​= 7). The top panel shows box plots showing the mean, median, and 75th to 25th percentiles of the threshold displacement required to evoke an action potential in these neurons.Mann–Whitney test. Figure 15g shows resting membrane potential values ​​recorded shortly after whole-cell current-clamp configuration was achieved from control and rat DRG neurons treated with MA (300 pM), n is denoted on the x-axis. Mann–Whitney test. Asterisks indicate values ​​significantly different from control (**p<0.01) and ns indicates not significantly different from control. Figures 16a–16g further show that MA decreases mechanically activated currents in rat DRG neurons. Figure 16a shows the current–voltage relationship of mechanocurrents in rat DRG neurons as determined by whole-cell membrane clamp experiments. Circles are mean + SD. n = 3. Figure 16b shows the inactivation time constant of peak displacement-evoked currents in control and rat DRG neurons treated with MA (300 μM for 18 h). Bars are mean ± SD. n is denoted on the x -axis. Unpaired t -test. Figure 16c shows representative current-clamp recordings of membrane potential changes evoked by a train of mechanical pulses from control and rat DRG neurons treated with MA.Figure 16d shows the number of action potentials evoked by sweeping versus mechanical indentation of control (n = 6) and MA-treated rat DRG neurons (n ​​= 6). Figure 16e shows the action potential amplitude measured from the resting potential to the maximal membrane potential of control and MA-treated rat DRG neurons (300 μM for 18 h). Bars are mean ± SD. n is denoted on the x -axis. Unpaired t -test. Figure 16f shows the normalized outward current densities evoked by step depolarization from a holding potential of −80 mV of control (n = 8) and MA-treated rat DRG neurons (n ​​= 6; 300 μM). Circles are mean ± SD.Figure 16g shows the normalized inward current densities evoked by step depolarization from a holding potential of -80 mV in control (n = 8) and MA-treated DRG neurons (n ​​= 6; 300 pM). Circles are the mean ± SD. Asterisks indicate values ​​significantly different from control (**p<0.01), and ns indicates not significantly different from control. Figures 17a–17d show that an omega-3-enriched diet decreases PIEZO2 inactivation time constants in mouse DRG neurons. Figure 17a shows representative PIEZO2 currents evoked by mechanical stimulation (at -60 mV) of DRG neurons dissected from WT mice fed control or omega-3-enriched diets. Figure 17b shows the maximal displacement-evoked PIEZO2 inactivation time constant of DRG neurons dissected from WT mice fed control or omega-3-enriched diets. Bars are mean + SD. n is denoted on the x -axis. Unpaired t -test. Figure 17c shows the maximal displacement-evoked current densities of DRG neurons dissected from control and omega-3-enriched diets. Bars are mean ± SD. n is denoted on the x -axis. Unpaired t-test.The box plots in Figure 17d show the mean, median, and 75th to 25th percentiles of the displacement thresholds required to evoke mechanical currents from DRG neurons dissected from the control and omega-3-enriched diets, n is denoted on the x -axis. Unpaired t test with Welch's correction. Asterisks indicate values ​​significantly different from the control (*p<0.05) and ns indicates values ​​that are not significantly different from the control. Figures 18a–18d show that eicosapentaenoic acid (EPA) supplementation decreases PIEZO2 inactivation time constants in rat DRG neurons. Figure 18a shows representative whole-cell membrane-clamp traces of mechanically activated currents from control and rat DRG neurons treated with EPA (200 μM for 18 h). Figure 18b shows the maximally displaced PIEZO2 inactivation time constants from control and rat DRG neurons treated with EPA (200 μM for 18 h). Bars are mean ± SD. n is denoted on the x -axis. Unpaired t -test. Figure 18c shows the maximally displaced current densities of control and rat DRG neurons treated with EPA (200 μM for 18 h). Bars are mean ± SD. n is denoted on the x-axis. Unpaired t test.The box plots in Figure 18d show the mean, median, and 75th to 25th percentiles of the displacement thresholds required to mechanically evoke control and rat DRG neurons treated with EPA (200 pM for 18 h), n is denoted on the x -axis. Unpaired t -test with Welch’s correction. Asterisks indicate values ​​significantly different from control (**p<0.01), and ns indicates values ​​that are not significantly different from control. Figures 19a–19c show that EPA supplementation abrogates the phenotype of PIEZO2 arthrogryposis mutants. Figure 19a shows a ribbon representation of the mouse PIEZO2 monomer highlighting the mutations that cause arthrogryposis in humans. Figure 19b shows representative normalized macroscopic currents (at –60 mV) evoked by the maximal displacement of N2A cells transfected with P1EZO2 arthrogryposis mutants S2691R and E2727del with and without EPA supplementation (left and right, respectively). Figure 19c shows the PIEZO2 inactivation time constants evoked by the maximal displacement of S2691R and E2727del arthrogryposis mutants with and without EPA supplementation. Bars are mean + SD. Unpaired t-test with Welch’s correction. Asterisks indicate values ​​significantly different from the control (**p<0.01 and ***p<0.001) and ns indicates not significantly different from the control.Figures 20a-201 show that EPA supplementation decreases the inactivation time constants of mouse PIEZO2 currents heterologously expressed in cells. Figure 20a shows representative whole-cell membrane-clamp recordings of control and PIEZO V2-transfected NZA1^21'1^ cells treated with EPA (200 μM; 18 h). Figure 20b shows the peak-shift-elicited inactivation time constants of control and PIEZO V2-transfected NZA1^21'1^ cells treated with EPA (200 μM; 18 h). Bars are mean + SD. n is denoted on the x -axis. Unpaired t -test. The box plots in Figure 20c show the mean, median, and 75th to 25th percentiles of the displacement thresholds required to evoke currents in control and PIEZO V2-transfected N2A / '?zh / cells treated with EPA (200 pM; 18 h). n is denoted on the x -axis. Unpaired t test.Figure 20d shows the maximal displacement-evoked current densities of control and PIEZO V2-transfected N2A / ''zo / / · cells treated with EPA (200 pM; 18 h). Bars are mean ± SD. n is denoted on the x -axis. Mann-Whitney test. Figure 20e shows representative whole-cell membrane-clamp recordings of control and PIEZO V16-transfected N2Ap'ez / _ / · cells treated with EPA (200 pM; 18 h). Figure 20f shows the maximal displacement-evoked inactivation time constants of control and PIEZO VI6-transfected NZA1''^01'1'' cells treated with EPA (200 pM; 18 h). Bars are mean ± SD. n is denoted on the x -axis. Unpaired t -test.The box plots in Figure 20g i? LRRnn / zznz / E / YiAi show the mean, median, and 75th to 25th percentiles of the displacement thresholds required to evoke currents in control and PIEZO V16-transfected N2Añezo77' cells treated with EPA (200 μM; 18 h). n is denoted on the x -axis. Unpaired t -test. Figure 20h shows the current densities evoked by maximal displacement in control and PIEZO VI6-transfected ^A^'277' cells treated with EPA (200 μM; 18 h). Bars are mean ± SD. n is denoted on the x -axis. Mann-Whitney test. Figure 20i shows representative whole-cell membrane binding recordings of control and PIEZO V14-transfected cells treated with EPA (200 μM; 18 h). Figure. 20j shows the maximal shift-evoked inactivation time constants of control and PIEZO V14-transfected ^A^2077' cells treated with EPA (200 μM; 18 h). Bars are mean + SD. n is denoted on the x -axis. Unpaired t -test. The box plots in Figure 20k show the mean, median, and 75th to 25th percentiles of the shift thresholds required to elicit currents in control and PIEZO V14-transfected ^A^2077' cells treated with EPA (200 μM; 18 h). n is denoted on the x -axis. Unpaired t -test. Figure 201 shows the peak displacement-evoked current densities of control and PIEZO V14-transfected N2Aftí'2°77 cells treated with EPA (200 μM; 18 h). Bars are mean ± SD. n is denoted on the x -axis. Mann-Whitney test. An asterisk indicates values ​​significantly different from the control (***p<0.001) and nsindicates that it is not significantly different from the control. Figures 21a–21d show that a combination of margaric and eicosapentaenoic acids (MA and EPA, respectively) decreases PIEZO2 currents and enhances inactivation in cultured mouse DRG neurons. Figure 21a shows representative whole-cell membrane-clamp recordings evoked by mechanical stimulation (at –60 mV) of PIEZO2 currents from control (left) and DRG neurons treated with MA (300 μM) + EPA (200 pM) (right). Figure 21b shows the maximally displaced PIEZO2 current densities of control and DRG neurons treated with MA (300 μM) + EPA (200 pM). Bars are mean ± SD. Figure 21c shows the shift thresholds required to evoke PIEZO2 currents from control and DRG neurons treated with MA (300 μM) + EPA (200 pM).Box plots show the mean (square), median (bisecting line), box boundaries (75th to 25th percentiles), outlier range with a coefficient of 1.5 (whiskers), and minimum and maximum data points. Figure 2Id shows the PIEZO2 inactivation time constants elicited by the i? LRRnn / zznz / E / YiAi peak displacement of control and DRG neurons treated with MA (300 μM) + EPA (200 pM). Bars are mean ± SD. n is denoted on the x -axis. P values ​​are denoted above the bars and boxes. The features described above and others will be appreciated and understood by those skilled in the art from the following detailed description, figures and appended claims. DETAILED DESCRIPTION The inventors previously explored how fatty acids influence mechanotransduction. When enriched in the plasma membrane, the esterified saturated fatty acid margaric acid (MA; heptadecanoic acid; 07:0) inhibits PIEZO1 channels by increasing the structural order and stiffness of the membrane, thereby raising the mechanical threshold required to activate the channel. Given PIEZO2's overall similarity to PIEZO1, the inventors reasoned that MA might also decrease PIEZO2 function and thus have therapeutic potential. However, unlike PIEZO1, which can be activated only by changes in membrane tension, PIEZO2 requires an intact cytoskeleton for normal function, as it can thus far only be activated in whole-cell or cell-attached membrane-tethered configurations. Thus, whether MA can efficiently modulate and decrease PIEZO2 activity remains to be determined. As shown in the present disclosure, the inventors use electrophysiological and behavioral approaches to determine that MA decreases PIEZO2 function under normal and inflammatory conditions. The inventors discovered that MA potently decreases PIEZO2 currents in a wide range of cell types, from mice and rats to humans, by increasing the mechanical stimuli required to activate the channel. Notably, MA supplementation combined with treatment with latrunculin A (i.e., a toxin that disrupts actin polymerization), reveals that PIEZO2 mechanosensitivity relies on both plasma membrane and cytoskeletal elements. Results from a PIEZO2-PIEZ01 chimera show that the PIEZO2 bundle, a large intracellular domain that runs parallel to the membrane and is thought to be critical for force sensing, dampens the effect of the membrane on Piezo2 activation.Importantly, the inventors determined that in dorsal root ganglion (DRG) neurons, MA efficiently reduces action potential firing evoked by mechanical stimuli but not by i? LRRnn / zznz / E / YiAi current injection, suggesting that MA could attenuate tactile responses in vivo. Furthermore, MA decreases PIEZO2 currents that are potentiated by the proalgesic agent bradykinin, indicating that it could be particularly useful for reducing tactile responses that are increased during inflammation. The inventors discovered that MA selectively decreases the increased tactile responses seen during a mouse model of repeated injury without affecting thermal responses. In one aspect, a method for treating pain comprises administering to a subject in need of pain treatment a pharmaceutical composition comprising a therapeutically effective amount of margaric acid. Illustrative types of pain treated include inflammatory pain, pain due to nerve injury, neuropathic pain, chronic pain, intractable cancer pain, complex regional pain syndrome, surgical or post-surgical pain, dental pain, pain resulting from skin injury, low back pain, headaches, migraines, allodynia, and hyperalgesia. In certain cases, pain is chronic. In other cases, pain is acute. Pain may be mild or severe. Illustrative pain indications include treatment or prophylaxis of surgical or postsurgical pain for various surgical procedures including amputation, post-cardiac surgery, dental pain / tooth extraction, pain resulting from cancer, muscle pain, mastalgia, pain resulting from dermal injuries, low back pain, headaches of various etiologies including migraine, menstrual cramps, tactile allodynia, and hyperalgesia. Pain may be somatogenic (both nociceptive and neuropathic), acute, and / or chronic. Peripheral neuropathies that may be treated with margaric acid include mononeuropathies, multiple mononeuropathies, and polyneuropathies, including axonal and demyelinating neuropathies. Both sensory and motor neuropathies are included. Neuropathy or neuropathic pain may be associated with a number of peripheral neuropathies of various etiologies, including, but not limited to: trauma-induced neuropathies,which include those caused by physical injuries (such as blunt trauma, abrasions, or burns) or pathological conditions, physical damage to the brain, physical damage to the spinal cord, or stroke that is associated with brain damage; neurological disorders that are associated with neurodegeneration; and postsurgical neuropathies and neuropathic pain (such as that of shingles, diabetes, and the like); infectious and viral neuropathies, including those caused by leprosy, Lyme disease, a herpes virus (and more particularly by the herpes zoster virus, which can cause postherpetic neuralgia), human immunodeficiency virus (HIV, which can cause HIV neuropathy), 17i? LRRnn / zznz / E / YiAi or papillomaviruses, or any other pathogen-induced nerve damage; Toxin-induced neuropathies (including, but not limited to, alcoholism-induced neuropathies, vitamin B6 intoxication, hexacarbon intoxication,amiodarone, chloramphenicol, disulfuram, isoniazid, gold, lithium, metronidazole, misonidazole, nitrofurantoin); drug-induced neuropathies, including therapeutic drug-induced neuropathy, particularly a) chemotherapy-induced neuropathies caused by anticancer agents such as taxol, taxotere, cisplatin, nocodazole, vincristine, vindesine, and vinblastine, and b) antiviral neuropathies caused by antiviral agents such as ddl, DDC, d4T, foscamet, dapsone, metronidazole, and isoniazid); vitamin deficiency-induced neuropathies, including those resulting from vitamin B12 deficiency, vitamin B6 deficiency, and vitamin E deficiency); hereditary neuropathy (including, but not limited to, Friedreich's ataxia, familial amyloid polyneuropathy, Tangier disease,Fabry disease; diabetic neuropathy and neuropathy due to metabolic disorders such as renal failure and hypothyroidism; neuropathy secondary to tumor infiltration; autoimmune neuropathies, including those resulting from Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, monoclonal gammopathy of undetermined significance and polyneuropathy, and multiple sclerosis; other neuropathies and neuropathic pain syndromes including inflammation-induced nerve damage, neurodegeneration, post-traumatic neuralgia, central neuropathic pain syndromes such as phantom limb pain, pain, complex regional pain syndromes (including, but not limited to, reflex sympathetic dystrophy, causalgia), pain associated with neoplasia, vasculitic / angiopathic neuropathy and sciatica, and idiopathic neuropathies. Neuropathic pain can manifest as allodynia, hyperalgesic pain, or phantom pain. In other forms, neuropathy can lead to a loss of pain sensitivity. There are three types of allodynia. Tactile allodynia (also called static) is pain triggered by touch, such as when clothing is placed against the skin or when a person lightly touches the arm. Mechanical allodynia (also called dynamic) is pain triggered by movement across the skin, such as drying oneself with a towel or the rubbing of bed sheets against the skin. Thermal allodynia is pain triggered by heat or cold that does not damage tissue. Patients with allodynia experience pain in response to stimuli that are generally considered harmless. Fibromyalgia is a disease that has chronic, unbearable systemic pain as its central symptom, accompanied by various comorbid symptoms such as insomnia, a feeling of systemic fatigue, depressive symptoms, and the like. Fibromyalgia is often accompanied by tactile allodynia. Fibromyalgia can also be accompanied by mechanical allodynia and thermal allodynia.Additional medical conditions that are associated with allodynia include chronic inflammation, migraines, trigeminal neuralgia, postherpetic neuralgia, peripheral neuropathy, diabetic neuropathic pain, chronic fatigue syndrome, complex regional pain syndrome, and the like. Inflammatory pain includes inflammatory joint pain, inflammatory musculoskeletal pain, pain due to injury, arthritis pain, and complex regional pain syndrome. The agent may be administered via any route customarily used to administer a pain management medicament, including, but not limited to, oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intra-articular, and intramedullary), transmucosal (including nasal), transdermal, and topical (including dermal, buccal, sublingual, and intraocular) routes. Intravenous delivery, for example, may occur via bolus injection or infusion; the infusion may be administered over a period ranging from less than one minute to several hours or continuously. In certain embodiments, a course of treatment will involve administration by a combination of routes. The pharmaceutical compositions may include various pharmaceutically acceptable additives including, but not limited to, carriers, excipients, binders, stabilizers, antimicrobial agents, antioxidants, diluents and / or supports. As used herein, topical administration means non-systemic administration. This includes applying a compound described herein externally to the epidermis or instilling such a compound into the ear, eye, or nose, such that the compound does not significantly enter the bloodstream. In contrast, systemic administration refers to oral, intravenous, intraperitoneal, and intramuscular administration. In one aspect, topical or transdermal dosage of margaric acid provides 0.1 to 20 mg / kg of margaric acid. Formulations for transdermal administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of pain, such as liquids, gels, lotions, creams, ointments, or pastes. Formulations for transdermal administration may include excipients to solubilize the margaric acid. Drops suitable for administration to the eye, ear, or nose may also be employed as topical formulations. Margaric acid for transdermal or topical administration may comprise, for example, 0.01% to 10% w / w (by weight), 0.2-10% by weight, or 0.5-25% by weight of the formulation. Topical patches are topical formulations configured to deliver an active agent locally or transdermally to a subject when applied topically to a subject's skin surface. The formulations may include two or more layers, where the two or more layers may include at least an adhesive matrix and a backing. Formulations of the compounds described herein, suitable for oral administration, may be presented as discrete units, such as capsules, cachets, or tablets each containing a predetermined amount of the active ingredient, in powder or granule form; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion. Pharmaceutical preparations that can be used orally include tablets and capsules, such as gelatin capsules and soft capsules, as are known in the art. Tablets may be manufactured by compression or molding, optionally with one or more additional ingredients. Tablets may be prepared by compressing the active ingredient in a suitable machine in a free-flowing form, such as powder or granules, optionally mixed with binders, inert diluents, or lubricants, surface-active or dispersing agents. Molded tablets may be manufactured by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Injection formulations may be presented in unit dosage form, for example, in ampoules or multi-dose containers, with the addition of a preservative. The compositions may take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain excipients such as suspending, stabilizing, and / or dispersing agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and bottles, and may be stored in powder form or in freeze-dried (lyophilized) form requiring only the addition of the sterile liquid carrier, for example, sterile pyrogen-free saline or water, immediately prior to use. In addition to the formulations described above, the compounds may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. i? LRRnn / zznz / E / YiAi In one aspect, a transdermal pharmaceutical composition comprises margaric acid and a pharmaceutically acceptable excipient. In another aspect, a pain treatment composition comprises margaric acid, eicosapentaenoic acid, and a pharmaceutically acceptable excipient. Eicosapentaenoic acid (EPA; 20:5) is a dietary long-chain omega-3 fatty acid believed to be involved in reducing inflammation. When combined with MA, EPA is expected to further reduce tactile inflammation and tactile pain. In one aspect, a topical formulation comprises 0.49 to 0.55% by weight of margaric acid, 0.49 to 0.55% by weight of eicosapentaenoic acid, 25.05 to 27.69% by weight of a principal solvent, 23.77 to 26.27% of a cosolvent, and 45.19 to 49.95% by weight of a viscosity agent, all weights being based on the total weight of the topical formulation. In a further aspect, the principal solvent is sesame oil, the cosolvent is paraffin oil, and the viscosity agent is castor oil. The invention is further illustrated by the following non-limiting examples. EXAMPLES Methods Cell Culture and Electrophysiology: Piezol knockout mouse N2A cells (NZA^077') were a gift from Dr. Gary R. Lewin, the human Merkel cell carcinoma cell line (MCC13 cells; Cell Bank Australia accession number: CBA1338) was obtained from Sigma, and DRG neurons were obtained from sacrificed mice. ^A^27 7 cells were grown in Dulbecco's modified Eagle's medium (DMEM), 5% penicillin-streptomycin, and 10% fetal bovine serum (EBS); MCC13 cells were grown in RPM1 1640 (with 2 mM L-glutamine + 25 mM HEPES; Sigma), 5% penicillin-streptomycin, and 10% FBS; DRG neurons are cultured in DMEM, 1% penicillin-streptomycin, 1% MEM vitamin solution, 1% L-glutamine, and 10% horse serum. Prior to electrophysiological measurements, N2A, zo / / ·, MCC13, and DRG neurons are supplemented overnight (~18 h) with MA.For accumulation assays, cells are supplemented with 50 μM of MA every 24 h for 5 days. MA is obtained from Nu-Chek Prep, INC. Cultured cells are maintained at 37°C, 95% relative humidity, and 5% CO2. Rat DRG neurons (R8820N10) are obtained from Cell Applications, INC. Neurons are thawed and cultured according to the manufacturer’s protocol and used between days 3-5 post-thaw. i? LRRnn / zznz / E / YiAi For whole-cell recordings, the bath solution contains 140 mM NaCl, 6 mM KCl, 2 mM CaCl, 1 mM MgCl, 10 mM glucose, and 10 mM HEPES (pH 7.4; 300 mOsm). The pipette solution for voltage-clamp recordings contains: 140 mM CsCl, 5 mM EGTA, 1 mM CaCl, 1 mM MgCl, and 10 mM HEPES (pH 7.2); and for current-clamp recordings: 140 mM KCl, 6 mM NaCl, 2 mM CaCl, 1 mM MgCl, 10 mM glucose, and 10 mM HEPES (pH 7.4; 300 mOsm); MA and bradykinin acetate salt (Sigma) perfused during the experiments are dissolved in the bath solution to a final concentration of 300 μM for 2 min and 1 pM for 5 min, respectively; and for long-exposure experiments, the culture medium is supplemented with bradykinin and added to the cells 18–24 h before recording.For cytoskeletal disruption experiments, N2Aβ“'ΖΛΛ is incubated in media supplemented with 1 pM latrunculin A (Cayman Chemicals) during pre-Ih recordings. Pipettes are made of borosilicate glass (Sutter Instruments) and fire-polished to a resistance between 3 and 5 MW before use. During mechanical stimulation, currents are recorded at a constant voltage (-60 mV, voltage clamp) and voltages are recorded without injecting current (current clamp). Both variables are sampled at 100 kHz and low-pass filtered at 10 kHz using a MultiClamp™ 700 B amplifier and Clampex (Molecular Devices, LLC). To measure voltage-gated currents, a square-pulse protocol consisting of 20 mV increments of 40 ms starting from -80 mV in 500 ms intervals with P / 4 subtraction is used; and to record action potentials evoked by current injection, they are injected in increments of 20 pA of 40 ms in 500 ms intervals. In both cases, variables are sampled at 20 kHz and low-pass filtered at 10 kHz.Leakage currents before mechanical stimulations are subtracted from the current traces, and the data are digitally filtered at 2 kHz using ClampFit (Molecular Devices, LLC). Recordings with leakage currents >200 pA, with access resistance >10 MW, and cells whose giant seals cannot withstand at least six consecutive stages of mechanical stimulation are excluded from the analysis. Mechanical stimulation: For indentation assays, N2Aft£,zo / / ·, MCC13 cells, DRG neurons, and human IPSC374-derived neurons are mechanically stimulated with a heat-polished blunt glass pipette (3–4 pm) driven by a piezoelectric servo drive (E625, Physilc Instrumente). The blunt pipette is mounted on a micromanipulator at a -45° angle and positioned 3–4 pm above the cells without indenting them. Displacement measurements are obtained with a square pulse protocol consisting of indentation steps of 1 pm, each lasting 200 ms with a 2 ms ramp at 10 s intervals. The threshold of the mechanoactivated currents for each experiment is defined as the indentation stage that evokes the first current deviation from the baseline.For current-clamp experiments, the mechanical threshold is defined as the indentation stage that elicits the first action potential. For pulse train assays, 13-s sweeps with a 1 Hz train rate of 200-ms square pulses are used. Subsequent sweeps are in 1-pm increments. Only cells that did not detach during the stimulation protocols are included in the analysis. The piezoelectric servo driver is automated using a MultiClamp™ 700B amplifier via Clampex (Molecular Devices, LLC). Transfection of N2A / V;í' / 7? cells N2A / 7'277 cells are co-transfected with 75–200 ng / ml-l of the mm Piezo2 variants (2, 14, and 16), or 1 ng / ml-l of the PIEZO2-PIEZO1 bundle chimera cloned into pcDNA3.1 and GFP391 pMO; by using Lipofectamine® 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions and recorded 48 h later. Fatty acids are supplemented 18–24 h prior to recording unless otherwise stalled. Primary DRG Neuron Culture: Primary DRG neuron cultures are obtained from 8-12 week old C57BL / 6 male mice. Mice are anesthetized with isoflurane and then sacrificed by cervical dislocation. DRGs are dissected and kept on ice in Hank’s Balanced Salt Solution IX (BBSS without CaChy MgCb). DRGs are then incubated in 1 mg / ml collagenase B (Sigma) in HBSS at 37°C and 5% CO2, and after 1 hour they are dissociated in serum-free medium. The cell suspension solution is centrifuged for 8 min at 800 rpm. The resulting pellet is resuspended in complete DMEM medium containing 1% penicillin-streptomycin, 1% MEM 402 vitamin solution, 1% L-glutamine, and 10% horse serum. Cells are grown on coverslips pretreated with poly-L-lysine. All cultured neurons are used after 18–24 h. Mouse behavior: MA solution: MA is prepared in sterile Dulbecco's phosphate-buffered saline solution, containing 70 g / l fatty acid-free bovine serum albumin (BSA; Sigma), and MA to a final concentration of 5 mM. The solution is filtered to remove undissolved materials. Subjects: For the experiment, 8-12 week old male and female C57B16 / J mice (N=12; control n=6; males=3, females=3) MA (n=6; males=3, females=3) are used for 23 i? LRAnn / zznz / E / YiAi experiment. Mice are housed under a 12-hour light / dark cycle and are allowed access to food and water ad libitum. Treatment: Animals are divided into two groups (control and MA) and given injections in the sole of the left hind paw for 7 days prior to behavioral assessment. Injections are administered as follows: The mouse is restrained by its tail, paw, and neck above the shoulders. The compound is delivered using a 3 / 10 cc insulin syringe into the plantar surface of each mouse's hind paw. The beveled needle is inserted at a 35–40° angle, and 20–50 ml of the compound is injected subcutaneously. The needle is held in place for several seconds before being removed and the animal is returned to its home cage. All females are handled and treated before males to reduce stress. Before all behavioral tests, animals are habituated to the test apparatus for ~30 min. To induce acute inflammation, AITC (lmM) is applied to the paw using a small paintbrush. Mechanical threshold (Von Frey test): Punctate mechanical allodynia is measured using Von Frey monofilaments (Stocking, Inc.) of various grams of force (0.008–1 g). Animals are placed in small enclosures on a wire mesh, and the sole of the hindpaw is pricked with a filament, and the responses are recorded. Withdrawal responses are recorded when the mesh and sole of the hindpaw are pricked with a filament, and the responses are recorded. Withdrawal responses are recorded when the animal lifts, shakes, or licks its paw in response to the stimulus presentation. If the animal makes no movement in response to the stimulus presentation, no response is indicated. Each filament is pressed against the sole until the fiber bends slightly. The amount of force required to bend the fiber depends on the thickness of each monofilament.A response is indicated by five withdrawal responses to a filament, the test is stopped for each subject when the withdrawal to any particular filament is ten. Mechanical pain (pinprick): Animals are placed in small enclosures on a wire mesh screen and the sole of the hindpaw is punctured with a needle (25G 5 / 8”) at a 45° angle. Responses are recorded; stimulus presentations occur ten times per hindpaw. Thermal threshold (Hargreaves test): Animals are placed in small enclosures on a glass plate heated to 32°C (IITC). A focused radiant heat light source is applied to the sole of the hindpaw. The latency for each rodent to withdraw its paw is recorded with three stimulus presentations administered per paw. The mouse is restrained by its tail, paw (LRAnn / zznz / E / YiAi), and the nape of the neck above the shoulders. The compound is delivered using a 3 / 10 cc insulin syringe into the plantar surface of the hindpaw of each mouse. The beveled needle is inserted at a 35–40° angle, and 20–50 μA of the compound is injected subcutaneously. The needle is held in place for several seconds before being removed and the animal returned to its home cage. All females are handled and treated before males to reduce stress. Human iPSC-derived neurons: To generate human peripheral sensory neuron cultures, a version of the healthy control WTC11 iPSC line is used. This line is pre-engineered to harbor a doxycycline-inducible NGN2-BRN3A construct that enables rapid and efficient sensory neuron differentiation. Undifferentiated iPSCs are maintained in E8 flex medium (Invitrogen) on Matrigel© (Corning)-coated polystyrene dishes. The medium is changed every 1–3 days, and cells are passaged every 4–7 days with Accutase® (Invitrogen) and seeded overnight with 10 pM of the ROCK inhibitor Y-27632 (Tocris). For sensory neuron differentiation, iPSCs are seeded at 20,000 cells (cm2)-l in neural differentiation medium (NDM) on Matrigel®-coated dishes. Cells are then replated after 48 hours at 50,000 cells·(cm2)1 on plates coated with polyethyleneimine (Sigma-Aldrich) and laminin (Invitrogen).NDM consists of (all Invitrogen) DMEM / F12 1:1 and Neurobasal™ medium supplemented with N2, B27, and GlutaMAX™ at the manufacturer’s recommended dilution. 2 µg / ml of doxycycline (Clontech) is included in the medium for the duration of the culture, 10 µM Y-27632 is supplemented for the first 48 hours, and the following neurotrophic factors are added from day 8 onward at 10 ng / ml each (all R&D systems): BDNF, GDNF, β-NGF, and NT-3. Complete medium changes are performed every other day until after day 8, and then half-volume medium changes are performed every other day for the remaining time in culture. Before electrophysiological recording, a subset of plates is supplemented with 300 or 600 pM of AM for 18 hours or 50 pM of AM for 5 days. All recordings are made in neurons cultured for 14–16 days. Data analysis: Results are expressed as means + SD (unless otherwise indicated). Box plots represent the range between the 25th and 75th percentiles, the mean, median, and outliers with a coefficient of 1.5. Data are plotted using OriginPro (from OriginLab). The inactivation time constant t is obtained by fitting a single exponential function (1) between the peak current value and the end of the stimulus: i? LRRnn / zznz / E / YiAi i? LRRnn / zznz / E / YiAi where A = amplitude; t = time constant; and the displacement and constant C for each component i. The sigmoidal fit was performed using OriginPro with the following Boltzmann function: where A2 = final value; Ai = initial value; Xo = center; and dX = time constant. Statistical analyses were performed using GraphPad Instat 3 software. Individual tests are described in each of the figure legends. Example 1: Margaric acid inhibits PIEZO2 currents in N2A cells PIEZO2 channels are characterized for the first time in Neuron-2a (N2A) cells transfected using an electrically actuated glass piezoelectric probe. It was previously determined that N2A plasma membranes can be enriched with MA after overnight incubation and promote high bending stiffness, as determined by mass spectrometry and atomic force microscopy. Importantly, PIEZO1 is determined to display reduced activity in this membrane environment. To determine whether PIEZO2 can also be modulated by membrane mechanical properties, the V2 variant of Piezo2 is transfected and its mechanical currents are measured after supplementing the cellular media (i.e., cells in which the PIEZO1 gene has been deleted) with MA, ranging from 1–600 μM, overnight.MA is found to inhibit PIEZO2 currents in a concentration-dependent manner (Figures 1a, 1b and Figure 2a) with an IC50 = 190.6 + 14.7 μM (mean + SEM; Figure 1b). Furthermore, MA increases the threshold displacement required to elicit PIEZO2 currents by threefold compared to that in control cells (Figure 1c), without affecting the inactivation time constant (Figure 2b). MA concentrations greater than 100 μM are required to decrease PIEZO2 currents when supplementation is used overnight (Figure 1b). Fatty acids can accumulate when their intake is increased through diet, and it has been previously shown that MA can also accumulate at the plasma membrane when supplemented into the cellular medium for several days at low concentrations. Therefore, to inhibit PIEZO2 activity with lower doses of MA, a daily supplementation protocol was implemented. Indeed, supplementing cells with only 50 μM of MA over the course of four days reduced PIEZO2 currents by 65% ​​(Figures 1d, 1e). As seen with higher concentrations overnight, our serial low-dose MA supplementation increases the translocation threshold without altering PIEZO2 inactivation (Figure 1f and Figure 2c).Similar results are obtained by supplementing with 25 μM daily for eight days (Figures 2d–2g). This demonstrates that MA concentrations could be further reduced by prolonging the incubation time. Notably, MA also inhibits the activity of two other Piezo2 variants that are particularly abundant in the trigeminal ganglion (V14 and 16), indicating that it likely affects most of the alternatively spliced ​​isoforms of this channel (Figures 3a–3i). Together, our results demonstrate that MA inhibits PIEZO2 currents by increasing the mechanical threshold required for activation. Thus, like PIEZO1, PIEZO2 is less active on stiff membranes (>78 pN). Example 2: PIEZO2 function relies on the plasma membrane and cytoskeleton By comparing the activities of PIEZO1 and PIEZO2 under increasing concentrations of MA, the ICso for PIEZO1 is determined to be 28.3 ± 3.4 μM and for PIEZO2 is 190.6 ± 14.7 μM (mean 127 ± SEM; Figure 4a, Figures 5a-5d). Several lines of evidence suggest that PIEZO2 requires the cytoskeleton for activation, whereas PIEZO1 can be activated solely by inside-out patchy membrane tension. These distinct features may explain why ~7 times more MA is required to inhibit PIEZO2 channels than PIEZO1. We previously showed that disrupting actin filaments does not affect the bending stiffness of the plasma membrane of N2A cells that are not treated or enriched with MA.Therefore, to determine the contribution of the actin cytoskeleton to PIEZO2 activation, we treated MA-enriched cells with latrunculin A and compared their mechanically evoked responses with those of cells treated with MA alone. Treatment with latrunculin A results in a pronounced leftward shift in the MA dose-response profile for PIEZO2 (IC50= 75.4 ± 13.3 μM; mean ± SEM, Figure 4b) that is closer to that of Piezol (Figure 4a). On the other hand, the MA 137 dose-response profile of PIEZO1 is similar in control and latrunculin-treated cells (IC5o= 28.3 μM + 3.4 of control vs. 25.6 μM ± 8.4 of latrunculin-treated cells, mean 138 ± SEM; Figure 4c and Figures 5c, 5d), indicating that the mechanism of PIEZO1 current inhibition by MA is only plasma membrane-dependent.These results implicate the cytoskeleton as a key determinant of the differential responses of PIEZO2 and PIEZO1 to MA. Unlike PIEZO1, the effect of a rigid plasma membrane (i.e., MA-enriched 27i? LRRnn / zznz / E / YiAi) on PIEZO2 becomes more evident when the cytoskeleton is pharmacologically disrupted (Figures 4b, 4c). We next asked whether modifying intracellular regions of PIEZO2 (which likely interact with cytoskeletal elements) could enhance MA inhibition, similar to the effect seen with latrunculin A treatment. Both PIEZO1 and PIEZO2 contain a 90 A long intracellular helix called a bundle (i.e., connecting the transmembrane blades to the central pore), which we reasoned might also link the channels to the cytoskeleton. In particular, the sequence identity between PIEZO1 and P1EZO2 bundles is low (35%) and could therefore explain the different responses of these channels to MA inhibition.To test this hypothesis, a PIEZO2 chimera was designed by replacing its bundle with that of PIEZO1 (Figure 4d). The PIEZO2-PIEZO1 bundle chimera displays similar functional properties to PIEZO2, including the reversal potential (7.7 mV for PIEZO2 vs. 5.6 mV for the chimera) and the displacement threshold (6.27 ± 1.35 pm for PIEZO2 vs. 6.9 ± 0.8 pm for the chimera, mean ± SD; Figures 6a–6c). However, the inactivation time constant of the chimera is faster than that of PIEZO2 (7.59 ± 1.96 ms for PIEZO2 vs. 1.91 ± 0.46 ms for the chimera, mean ± SD; Figures 2c and 6d). Surprisingly, transfer of the PIEZO1 bundle to PIEZO2 results in channels that are much more sensitive to MA inhibition (100 μM overnight), similar to those observed for PIEZO1 (Figure 4e).This chimera requires a greater mechanical stimulus to open when expressed in cells supplemented with MA (100 μM overnight; Figure 6c) and this effect is not modulated by latrunculin A treatment (Figures 4c, 4f). Taken together, our results highlight that PIEZO2 mechanosensitivity relies on the synergy between plasma membrane mechanics and interaction with cytoskeletal elements. Example 3: Margaric acid decreases mechanical currents in sensory cells. Piezo2 is expressed in Merkel cells and their innervating afferents, where it has been shown to transduce skin indentation and whisker deflection into electrical signals. In view of the results described above in a heterologous expression system, we wondered whether MA could decrease PIEZO2 currents in cells that mediate tactile sensation. To this end, we measured the effect of MA on PIEZO2 activity in the human Merkel cell carcinoma cell line (MCC13) and acutely cultured mouse DRG neurons. Like dissociating Merkel cells, MCC13 displays mechanosensitive 28i LRRnn / zznz / E / YiAi currents with a range of inactivation kinetics (Figure 7a). These mechanical currents have been shown to be mediated by PIEZO2.As with our experiments using transiently transfected N2A cells, MA supplementation in MCC13 decreases endogenous PIEZO2 currents (Figures 7a, 7b) by increasing the displacement threshold (Figure 7c). Similarly, cultured mouse DRG neurons also exhibit mechanical currents with variable inactivation kinetics. However, in this case, only rapidly adapting currents (t < 10 ms) have been assigned to PIEZO2. Notably, MA supplementation decreases the current magnitude of all mechano-evoked currents of DRG neurons, including those known to be mediated by PIEZO2 (Figures 7d, 7e) by increasing the displacement threshold (Figure 7f). Taken together, these findings translate our heterologous expression results to show that MA decreases endogenous mechanical currents in diverse cell types known to be involved in mechanosensation.Example 4: Margaric acid decreases action potential activation evoked by mechanical stimuli. Touch detection relies on mechanosensitive ion channels expressed in sensory nerve terminals. These channels translate mechanical stimuli into electrical signals, depolarize neurons, and, in turn, generate action potentials that propagate to the central nervous system. PIEZO2 mediates a significant proportion of the mechanically activated excitatory currents in mouse DRG neurons. Since MA decreases mechanical currents (including those of PIEZO2), we sought to determine whether this saturated fatty acid would also affect the ability of DRG neurons to elicit mechanically activated action potentials.Indeed, we found that MA completely inhibited action potential generation in mouse DRG neurons when indentation steps were less than 12 pm (Figure 8a, left and middle panels). However, action potentials could be evoked in MA-treated neurons after using larger indentation steps [12 pm for MA (red steps) vs. 7–12 pm for control; Figure 8a, right panel and 8b]. Furthermore, stimulation of DRG neurons with a series of 1 Hz mechanical stimulus trains revealed that MA-enriched cells evoked fewer action potentials than control (<12 pm; Figure 8c), even at large indentation magnitudes (>12 pm; Figure 8d). Interestingly, MA-enriched neurons also displayed a progressive decrease in membrane potential as the number of indentation pulses progressed (Figure 8c, inset).This suggests that after repetitive stimulation, mechanosensitive channels are more difficult to open in neurons treated with MA. Overall, MA supplementation increases the mechanical threshold required for DRG neurons to fire action potentials. Mechanically driven depolarization in DRG neurons activates voltage-gated Na+ and K+ channels that are critical for generating action potentials. To determine whether MA affects the function of ion channels downstream of mechanical activation, voltage-gated currents were recorded in the presence or absence of MA. No significant differences were found in the amplitudes of the inward Na+ and outward K+ voltage-gated currents of control and MA-enriched DRG neurons (Figures 9a–9c). Furthermore, MA supplementation did not alter the membrane potential when measured in the whole-cell configuration (Figure 9d). Notably, no differences were found in action potential firing evoked by current injection between control and MA-enriched neurons (Figures 9e, 9f). Taken together, our results indicate that MA does not alter DRG neuronal electrical excitability, but rather specifically reduces action potential firing evoked by mechanical stimulation. Example 5: Margaric acid counteracts bradykinin sensitization of PIEZO2 Tissue damage is frequently accompanied by the accumulation of proalgesic inflammatory agents such as bradykinin, eicosanoids, and protons. These inflammatory molecules bind or interact with various membrane proteins, activate intracellular signaling cascades, and increase sensitivity to sensory stimuli, leading to allodynia or hyperalgesia. Mechanically evoked currents by PIEZO2 have been shown to be potentiated downstream of bradykinin beta 2 receptor activation in DRG neurons. Therefore, molecules that decrease PIEZO2 sensitization could be beneficial for treating mechanical allodynia. Given that MA significantly decreases PIEZO2 currents in DRG neurons (Figures 7d–7f), we wondered whether MA supplementation also decreases bradykinin-mediated PIEZO2 sensitization.Similar to previous reports, we find that acute bradykinin infusion sensitizes the mechanoactivated currents of DRG neurons (2.5-fold increase; Figure 10a, top and 10b). As predicted, MA supplementation decreases mechanical currents, even after bradykinin sensitization (Figure 10a, bottom). Surprisingly, the currents recorded in neurons supplemented with MA after administration of 30 i LRAnn / zznz / E / YiAi bradykinin (Figure 10a and Figure 11) closely resemble those of control DRG neurons (3.36 ± 1.68 pA / pF control vs. 3.64 ± 1.96 pA / pF bradykinin with MA, mean + SD; Figure 10b). Similar findings are observed with longer exposures to bradykinin. Overnight incubation with bradykinin enhances the magnitude of all mechanoevoked currents of DRG neurons (Figure 10c middle panel and lOd).However, combined overnight incubation with bradykinin and MA restored current densities to those of control neurons (Figures 10c, 10d). Together, these results support that enriching the plasma membrane with MA could counteract bradykinin-evoked mechanical sensitization by reducing mechanical currents to non-inflammatory levels. Therefore, MA is a promising molecule for decreasing mechanical hypersensitivity. Example 6: Margaric acid decreases ββ2-mediated mechanical allodynia Previous work from our group and others has shown that PIEZO2 is required for mechanical allodynia in both mice and humans. An important prediction from our data is that MA administration would protect against this type of pain. Acute subdermal injection of MA had no effect on baseline tactile responses (Figures 12a, 12b), likely because fatty acids require time to be incorporated and accumulate in membranes to modulate PIEZO2 function. Therefore, an experiment was designed to examine nociceptive responses after chronic exposure to MA (Figure 12a). Mice injected daily with a small amount of saline in the hindpaw displayed pronounced hypersensitivity to punctate touch within 7 days (a repeated injury model; Figure 12b). Surprisingly, mice failed to develop this type of heightened withdrawal response when MA was included in the injections (Figure 12b). Importantly, this type of mild injury or daily exposure to MA had no effect on thermal withdrawal latencies as measured by the Hargreaves radiant heat test (Figure 12c). Allyl isothiocyanate (AITC), the pungent compound in wasabi and mustard oil, elicits pain by activating nociceptors. AITC was topically applied to the left hindpaws of mice to induce heightened neuroinflammatory pain. Typically, AITC causes mice to immediately withdraw their hindpaws from even the lightest von Frey filaments 253 (<0.16 g; Figure 12d). However, mice treated with MA for 7 days responded with sensitivities similar to their baseline response from the previous week (Figure 12d).Together, these results provide proof of concept that reducing the function of 31 i? LRRnn / zznz / E / YiAi. Ρ1ΕΖΟ2 by MA is a feasible pharmacological approach to treat mechanical allodynia. Example 7: Margaric acid decreases mechanical currents in neurons derived from human iPSCs. Our previous results demonstrate that enriching the plasma membrane with MA has an inhibitory effect on mouse PIEZO2 function in vitro, ex vivo, and in vivo. These results have clear translational potential. We therefore explicitly tested the effect of MA in human sensory neurons. Recently, we developed a platform to robustly and reproducibly reprogram human induced pluripotent stem cells (iPSCs) into well-characterized neurons that have functional and transcriptional features indicative of low-threshold mechanoreceptors (Figure 13a). Notably, all of these in vitro-derived touch neurons have mechanically evoked currents that are entirely dependent on PIEZO2 expression. Overnight incubation of human iPSCs with MA (300 and 600 μM) significantly reduces endogenous PIEZO2 currents (Figures 13b and 13c).Furthermore, these results are confirmed by measuring mechanical currents of human PIEZO2 transfected into MA-supplemented ^A7'6,277 cells (Figures 14a-14b). Furthermore, supplementation of human IPSC270-derived neurons with 50 μM MA daily for 5 days (a strategy analogous to that used in mice) also significantly reduces PIEZO2 currents (Figures 13b-13c). As expected, MA increases the mechanical threshold required to activate the human channel, without altering the inactivation time constant, mirroring the results obtained with the murine ortholog (Figures 13d-13e and Figures 14c-14d). Similar to cultured mouse DRG neurons, MA does not change the voltage-activated currents of Na+ inward and K+ outward compared to neurons derived from control human iPSCs (Figures 13f-13h).These findings indicate that MA specifically affects mechanically activated currents while maintaining intact the electrical excitability of human sensory neurons. Since MA is commonly found in foods such as dairy fat, rye, and fish, it is speculated that including this fatty acid as a dietary supplement or topical ointment may be a strategy to alleviate mechanical allodynia in humans. Example 8: MA decreases mechanically activated currents in rat DRG neurons Figures 15a-15g and 16a-16g further show that MA decreases mechanically activated currents in rat DRG neurons. PIEZO2 is the essential transduction channel for tactile discrimination, vibration, and proprioception. Mice and humans lacking Piezo2 experience severe mechanosensory and proprioceptive deficits and do not develop tactile allodynia. Bradykinin, a proalgesic agent released during inflammation, potentiates PIEZO2 activity. Molecules that decrease PIEZO2 function could ameliorate tactile allodynia. Here, the dietary fatty acid margaric acid (MA) is found to decrease PIEZO2 function in a dose-dependent manner. Chimera analysis demonstrates that the PIEZO2 bundle is a key region tuning MA-mediated channel inhibition.MA reduces mechanically challenged neuronal action potential firing in mouse and rat neurons, bradykinin potentiation of PIEZO2, and PIEZO2 currents in tactile neurons derived from human induced pluripotent stem cells. Finally, we show that MA improves touch hypersensitivity after injury in mice. Our findings report on a natural product that inhibits PIEZO2 function, reveal a key region for channel inhibition, and provide evidence of new avenues for treating tactile allodynia and other forms of pain. Example 9: Combination of MA and EPA As shown in Figures 17a-17d, an omega-3-enriched diet decreases PIEZO2 inactivation time constants in mouse DRG neurons. As shown in Figures 18a-18d, eicosapentaenoic acid (EPA) supplementation decreases PIEZO2 inactivation time constants in rat DRG neurons. As shown in Figures 19a-19c, EPA supplementation abrogates the phenotype of PIEZO2 arthrogryposis mutations. As shown in Figures 20a-201, EPA supplementation decreases the inactivation time constants of mouse PIEZO2 currents that are heterologously expressed in N2Añí,zo / A cells. Example 10: MA and EPA have synergistic effects on PIEZO2 function in mouse DRG neurons. MA is previously shown to reduce PIEZO2 currents in mouse and rat dorsal root ganglia neurons, a human Merkel cell carcinoma cell line (MCC13), and human induced pluripotent stem cells (iPSCs) derived from touch receptor neurons. Importantly, several human diseases implicated in Piezo2 originate from increased PIEZO2 function due to decreased inactivation of currents. Figures 21a-21d demonstrate that the combination of MA and EPA impairs PIEZO2 function and affects two of its inherent features: 1) reducing currents while increasing the displacement threshold, and 2) potentiating its inactivation. As proposed above, individual or combined fatty acids will be used to develop different ointments / lotions formulations to counteract mechanical allodynia. Example 11: Illustrative topical formulations Tables 1 and 2 provide illustrative MA formulations. The process is provided in Table 3: i? LRRnn / zznz / E / YiAi Table 1: First illustrative formulation Preparation Date Monday, January 25, 2021 Active Ingredient Target Ingredient Name % Theoretical (w / w), ± 5% % Actual (w / w), ± 5% API-1 Heptadecanoic Acid (MA) 0.5 0.52% API-2 Eicosapentaenoic Acid (EPA) 0.5 0.52% Primary Solvent Sesame Oil 25 26.37% Cosolvent Paraffin Oil 25 25.02% Viscosity Agent Castor Oil QS @ 100 47.57% Total 100.00 Table 2: Second illustrative formulation Preparation Date Monday, January 25, 2021 Placebo Ingredient Target Ingredient Name % Theoretical (w / w), ± 5% % Actual (w / w), ± 5% API-I Heptadecanoic Acid (MA) 0 0.00% API-2 Eicosapentaenoic Acid (EPA) 0 0.00% Primary Solvent Sesame Oil 25 25.20% Cosolvent Paraffin Oil 25 24.72% Viscosity Agent Castor Oil QS a 100 50.08% Total 100.00 Table 3: Processing 1. Turn on the water bath to a minimum of 60°C. 2. Weigh the APIs and heat in the water bath. Ensure the API dissolves before the next step. 3. Weigh and add the sesame oil. Vortex to form a uniform mixture. Keep heating._________________________________________________________________________________ 4. Weigh and add paraffin oil. Vortex to form a uniform mixture. Keep heating._________________________________________________________________________________ 5. Weigh and add castor oil. Vortex to form a uniform mixture. Keep heating._________________________________________________________________________________ 6. Stop heating after 5 minutes. Allow the mixture to cool to room temperature._______________________________________________________________________________ 7. Store at room temperature (25 °C) until use Discussion Mechanosensory ion channels are essential as they allow us to detect innocuous, pleasurable, alarming, or painful stimuli. PIEZO2 has emerged as the primary molecular detector for specific aspects of light touch (vibration detection and tactile discrimination) through its expression in specialized epithelial cells (Merkel cells) and peripheral sensory neurons. Importantly, PIEZO2 is also essential for the experience of touch-evoked pain following injury or under chronic inflammation, a common condition known as tactile allodynia that remains difficult to treat. Specifically, proalgesic agents (such as bradykinin) produced in response to tissue injury potentiate the PIEZO2 response. Ideally, novel treatment approaches will be developed to specifically counteract this type of pain without affecting normal tactile function.In the current study, we demonstrate that the application of margaric acid, a natural product found in various food sources such as dairy and lamb fat, rye, and fish, effectively reduces Piezo2 function. Previously, we showed that MA can be efficiently enriched in various cell types and, as a consequence, alter the gating profile of PIEZO 1 channels by increasing the structural order and stiffness of the plasma membrane. Intriguingly, previous work suggests that PIEZO 1 and PIEZO2 have distinct gating mechanisms. While both are likely sensitive to membrane tension, only PIEZO2 function is dependent on an intact cytoskeleton. Less is known whether PIEZO2 gating also relies on the mechanical properties of the plasma membrane. Our data indicate that PIEZO2 gating is affected by membrane stiffness and that the 35i? LRRnn / zznz / E / YiAi bundle domain and cytoskeleton counteract the effect of membrane tension. This highlights that the bundle is a key region for tuning MA-mediated channel inhibition. Putting our data into a broader context, we favor the idea that PIEZO2 functions as part of a force-carrying center. In this model, PIEZO2 function is tightly controlled by a platform comprising the plasma membrane (fatty acid tails and polar head groups), stomatin-like proteins, cytoskeletal elements (actin and microtubules), and extracellular tethers (e.g., focal adhesions). How selective is MA for mechanosensing? Our data show that MA reduces the ability of DRG neurons to fire action potentials upon mechanical stimulation without affecting the membrane potential, current-evoked action potentials, and voltage-gated currents triggered by Na+ inward and K+ outward.Similarly, MA reduces PIEZO2 mechanical activation while leaving other ionic currents intact in iPSC-derived neurons. Furthermore, subcutaneous injections of MA did not affect the ability of mice to detect thermal stimuli. These results strongly support that this dietary fatty acid specifically reduces neuronal mechanical excitability. As such, MA appears to have many preferable properties over other identified mechanoreceptor antagonists, such as the tarantula peptide GsMTx-4, which failed to inhibit DRG neuron mechanocurrents. Furthermore, the conopeptide analog NMB-1 has been shown to inhibit only intermediate and slow-inactivating mechanosensitive currents, but not fast ones. Importantly, unlike peptide toxins, MA is able to inhibit all mechanosensitive currents in mouse and rat DRG neurons and in human iPSC-derived neurons. With the recent discovery that PIEZO2 is required for tactile allodynia in mice and humans, this channel has emerged as a promising target for treating inflammatory conditions. Our data show that MA is able to counteract bradykinin-evoked PIEZO2 potentiation by reducing mechanical currents to non-inflammatory levels. Therefore, MA appears to present a potential approach to decrease mechanical hypersensitivity following inflammation. Notably, under our behavioral conditions, MA selectively decreases tactile allodynia without notable effects on baseline touch sensitivity. Current experimental and clinical studies favor treatments that target peripheral sensory receptors while avoiding systemic delivery.Thanks to its high hydrophobicity, MA is a candidate for the development of topical lotion and / or cream treatments to help reduce inflammatory pain. The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed as covering both the singular and plural unless otherwise indicated herein or clearly contradicted by context. The terms first, second, etc., as used herein, are not intended to indicate any particular order, but are merely for convenience in indicating a plurality of, for example, layers. The terms comprising, having, including, and containing are to be construed as open-ended terms (i.e., meaning including, but not limited to) unless otherwise indicated.The mention of ranges of values ​​is simply intended to serve as a shorthand method for individually referring to each separate value that falls within the range, unless otherwise indicated herein, and each separate value is incorporated into the description as if it had been individually mentioned herein. The endpoints of all ranges are included within the range and may be independently combined. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context in any other way. The use of any and all examples or illustrative language (e.g., “as”) is intended to better illustrate the invention only and does not represent a limitation on the scope of the invention unless otherwise claimed.No language in the description should be construed as indicating any element not claimed as essential to the practice of the invention as used herein. Although the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for other elements without departing from the scope of the invention. Furthermore, various modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope of the invention. Therefore, it is considered that the invention is not limited to the particular embodiment described as the best considered mode of implementing this invention, but that the invention will include all embodiments that fall within the scope of the appended claims. Any combination of the elements described above in all their possible variations are included in the invention unless otherwise indicated in this description or otherwise clearly contradicted by the context.

Claims

1. A method for treating pain, comprising administering to a subject in need of pain treatment a pharmaceutical composition comprising a therapeutically effective amount of margaric acid.

2. The method of claim 1, wherein the pharmaceutical composition is a topical, transdermal, oral, transmucosal or parenteral composition.

3. The method of claim 1, wherein the pharmaceutical composition is a topical or transdermal composition for delivery through the skin.

4. The method of claim 3, wherein the topical or transdermal composition is a liquid, gel, lotion, cream, ointment, paste or a topical patch.

5. The method of claim 3, wherein the topical or transdermal dosage of margaric acid is from 0.1 to 20 mg / kg of margaric acid.

6. The method of claim 3, wherein the topical formulation is a lotion comprising margaric acid in an amount of 0.2-10% by weight of the total weight of the formulation.

7. The method of claim 1, wherein the pain is chronic or acute.

8. The method of claim 1, wherein the pain is mild to severe.

9. The method of claim 1, wherein pain is inflammatory pain, pain due to nerve injury, neuropathic pain, chronic pain, intractable cancer pain, complex regional pain syndrome, surgical or post-surgical pain, dental pain, pain resulting from a skin injury, lower back pain, headaches, migraine, or hyperalgesia.

10. The method of claim 9, wherein the pain is neuropathic pain.

11. The method of claim 10, wherein the neuropathic pain is allodynia. i? LRAnn / zznz / E / YiAi 12. The method of claim 11, wherein the allodynia is tactile allodynia, mechanical allodynia, or thermal allodynia.

13. The method of claim 12, wherein the subject suffers from fibromyalgia, chronic inflammation, migraines, trigeminal neuralgia, postherpetic neuralgia, peripheral neuropathy, diabetic neuropathic pain, chronic fatigue syndrome, or complex regional pain syndrome.

14. The method of claim 9, wherein the pain is inflammatory pain.

15. The method of claim 14, wherein inflammatory pain is inflammatory joint pain, inflammatory musculoskeletal pain, pain due to injury, arthritis pain, and complex regional pain syndrome.

16. The method of claim 1 wherein the pharmaceutical composition further comprises eicosapentaenoic acid.

17. A pharmaceutical composition comprising margaric acid and a pharmaceutically acceptable excipient.

18. The composition according to claim 17, in the form of a topical or transdermal composition.

19. A composition for the treatment of pain comprising margaric acid, eicosapentaenoic acid and a pharmaceutically acceptable excipient.

20. A topical formulation comprising 0.49 to 0.55 wt% margaric acid, 0.49 to 0.55 wt% eicosapentaenoic acid, 25.05 to 27.69 wt% a main solvent, 23.77 to 26.27 wt% a cosolvent, and 45.19 to 49.95 wt% a viscosity agent, i? LRRnn / zznz / E / YiAi All weights are based on the total weight of the topical formulation.

21. The topical formulation according to claim 20, wherein the main solvent is sesame oil, the co-solvent is paraffin oil, and the viscosity agent is castor oil.