Research On The Function Of Mechanically Sensitive Ion Channel Piezo In Promoting Aqueous Humor Outflow

Aqueous humor（AH）is produced by the ciliary process of the posterior chamber,and is effluxed to the Scleral veins.The homeostasis of aqueous humor production and outflow is critical for the maintenance of intraocular pressure（IOP）.Increased resistance to AC outflow is a key pathogenic cause of elevated intraocular pressure and primary open-angle glaucoma.Therefore,ensuring normal AC outflow is an important means to maintain intraocular pressure.The trabecular meshwork is a key tissue regulating the outflow of aqueous humor through the trabecular meshwork-Schlemm canal;the ciliary muscle is essential for the outflow of aqueous humor through the uveoscleral pathway.The previous research of the research group has proved that the mechanosensitive ion channel Piezo is highly expressed in the trabecular meshwork and ciliary muscle of the aqueous humor circulation pathway,and clarified that Piezo is involved in the aqueous humor circulation and changes the dynamic parameters of the aqueous humor.closely related.Therefore,clarifying the key role of Piezo in the regulation of aqueous humor outflow will be of great significance for the treatment of glaucoma in IOP lowering.Objective:Based on the self-developed Syringe-pump system,referring to the new theory of the i Perfusion system for the measurement of aqueous humor in the eyeball,the Goldman Equation was independently modified to explore the role of Piezo in sensing intraocular pressure,transmitting mechanical signals,and adjusting the dynamic parameters of aqueous humor,and then To clarify the role of Piezo in regulating the trabecular meshwork-Schlemm canal and uveoscleral aqueous humor outflow,and ultimately provide a theoretical basis for the development of new drugs for the treatment of glaucoma based on Piezo.Methods:Aiming at the regulation of uveoscleral aqueous humor outflow by Piezo,the expression of Piezo in C57 BL/6J uveoscleral aqueous humor outflow pathway was determined by immunofluorescence method;and mechanosensitive ion channel inhibitor Gs TMx4)to regulate Piezo function;by tracing method,the anterior chamber was perfused with PBS,TMRD-PBS,Yoda 1-TMRD-PBS,Gs TMx4-TMRD-PBS,with the help of tissue section technology and laser confocal microscopy,quantitatively analyze the fluorescence intensity in the uveoscleral aqueous humor outflow pathway tissue,and evaluate the effect of Piezo on uveoscleral aqueous humor outflow.Aiming at the regulation of trabecular meshwork-Schlemm canal aqueous outflow by Piezo,latex microspheres（beads）were injected into the anterior chamber of C57 BL/6J mice to simulate pathological high intraocular pressure,and the intraocular pressure was monitored in real time by Tonometer;,to accurately determine the aqueous humor flow coefficient（C_r）of the isolated eyeball with high intraocular pressure;perfuse Yoda1 to analyze the effect of Piezo on the outflow of aqueous humor from the trabecular meshwork-Schlemm canal;to analyze the normal cultured trabecular meshwork cells by LC-MS/MS analysis,20%cyclic tension treatment group,20%cyclic tension and Gs TMx4 treatment groups,to clarify the role of mechanosensitive channels（CMCs）in regulating the response of human trabecular meshwork cells（HTMCs）to mechanical force.Results:1.Piezo 1 and Piezo 2 are highly expressed in ciliary muscle and trabecular meshwork.2.The fluorescence intensity of tracer-TMRD in ciliary muscle decreased with the survival time after perfusion;Comparison results of fluorescence intensity at 30 min,60min and 90 min after perfusion showed that fluorescence intensity of experimental group（Yoda 1-TMRD-PBS）was significantly higher than that of control group（TMRD-PBS）.At 120 min after perfusion,there was no significant difference in fluorescence intensity among all groups.Within 60 min after perfusion,the fluorescence intensity of each group was as follows:Yoda 1-TMRD-PBS group>TMRD-PBS group>Gs TMx4-TMRD-PBS group>PBS group.These results suggest that activation of Piezo 1 promotes aqueous outflow rate in the uvealscleral pathway,and functional inhibition of mechanically sensitive ion channels including Piezo reduces aqueous outflow rate in the uvealscleral pathway.3.Injection of anterior chamber Beads caused high IOP,and the aqueous fluid flow coefficient decreased significantly compared with the normal group.By comparing the aqueous humor flow coefficient of PBS group and Yoda 1 group under pathological conditions:The fluid flow coefficient of the PBS group was 16.0±1.2 n L/min/mm Hg（n=9）,and that of the Yoda 1 group was 21.0±0.5 n L/min/mm Hg（n=11）.There was a significant difference between the two groups in the fluid flow coefficient C_r value.Moreover,the aqueous fluid flow coefficient of Yoda 1 group was 20%-45%higher than that of PBS group（4.0 n L/min/mm Hg）.4.Mechanosensitive channels regulate the response of human trabecular meshwork cells to mechanical changes,and their downstream signals include calcium signaling,cytoskeleton remodeling,autophagy,cell cycle and cell fate.Conclusion:1.Piezo is expressed in both the uveoscleral pathway and the trabecular meshwork-Schlemm pathway of aqueous humor outflow,and is highly expressed in the ciliary muscle and trabecular meshwork in key parts.2.Yoda 1 regulates the physiological function of ciliary muscle by activating the function of Piezo 1 and promotes the outflow of uveoscleral aqueous humor;Gs MTx4inhibits mechanosensitive ion channels including Piezo,thereby inhibiting the outflow of uveoscleral aqueous humor Aqueous drainage function of the pathway.This suggests that Piezo 1 plays an active role in the uveoscleral aqueous humor outflow pathway.3.In the pathological state,Yoda 1 stimulates Piezo 1,improves the outflow facility of aqueous humor in the trabecular meshwork,and promotes the outflow of aqueous humor through the trabecular meshwork-Schlemm canal pathway.It is speculated that by regulating Piezo,it may promote the outflow of aqueous humor in the trabecular meshwork of glaucoma patients,thereby reducing intraocular pressure.4.When HTMCs are mechanically stretched,differential proteins related to Ca²⁺homeostasis,autophagy,cell cycle and cell fate may be the key downstream signals for CMCs to adapt to mechanical force and promote aqueous humor outflow.5.By confirming the promoting effect of Piezo on the outflow of aqueous humor,it is suggested that Piezo may become a potential new target for the treatment of glaucoma.