| Gout is a recurrent inflammatory disease caused by the disorder of purine metabolism increased serum uric acid and the deposition of uric acid crystals(MSU)in synovium,cartilage and other tissues.At present,research on the occurrence,recurrence,diagnosis and treatment,drug screening of the disease is still insufficient.Voltage-gated sodium channels are nociceptive,and Tenascins may be involved in inflammation.Therefore,this paper attempts to detect the correlation between gout and Tenascins-Na~+channel,and provide a scientific basis for the discovery and design of drugs targeting gout-related channels.The main results are as follows:1.Behavioral testing of MSU gouty arthritis modelA model of gouty arthritis was established using 8-week-old male SD rats.The experimental group was injected with urate suspension and tendon protein C(TNC)solution,and the control group was injected with 0.9%normal saline.Changes of paw and mechanical pain threshold were detected at appropriate time before and after injection.The results showed that the right foot of rats in uric acid group showed a persistent inflammation such as red and swelling at 6 h,8 h,and 24 h after drug injection.And there was a significantly increasing of the volume of the right foot compared with the control.But there was no statistical difference between the TNC group and the control group.It indicated that the TNC with this dose may not cause inflammation in rats.In the mixed group of MSU and TNC the volume of the right foot was between the two drugs alone after injection 4-8 hours,and the degree of swelling was relieved compared with the MSU group.At the 4 h,8 h,24 h,and 48 h after drug injection,the mechanical pain threshold of the right foot of the urate group was significantly lower than that of the control.There was no statistical difference in the left foot mechanical pain threshold.These indicated that uric acid crystal could cause persistent mechanical pain sensitization on the test side of rats,but no mechanical pain sensitization on the opposite side of rats.The mechanical pain threshold of the right foot of the TNC group was not significantly different from that of the control group,but after 24 h,the mechanical pain threshold of the left foot of the rat showed a significant decrease,suggesting that TNC may cause the occurrence of contralateral mechanical hyperalgesia in rats.In the mixed group of MSU and TNC,after 4 h-8 h,the mechanical pain threshold of the right foot of the rat was between the two drugs alone,which was higher than that of the MSU group.There was no statistical difference in the left foot mechanical pain threshold,indicating TNC relieves the acute occurrence of gouty arthritis.2.Regulation of sodium Current by MSU/Tenascins/beta Subunit2.1 Effect of MSU on total sodium current in rat dorsal root ganglion(DRG)Incubation of acutely isolated rat DRG cells with different doses of urate solution after different times.The total sodium current was recorded under voltage clamp.The results showed that after incubation of DRG cells with 0.1μg MSU for 2 hours,the activation of sodium current was delayed and the peak current was decreased,which was significantly different from that in the blank group.After incubating DRG cells with 10μg MSU for 5 hours,the inactivation of sodium channel accelerated and the peak current decreased,which was significantly different from that of the blank group.2.2 Effect of MSU on sodium Current in ND7/23 CellsIncubation of ND7/23 cells with different doses of urate solution after different times,sodium current was recorded under voltage clamp.The results showed that the shorter the incubation time of MSU,the more it slowed the activation of sodium current and reduced the peak of sodium current amplitude at the high dose of 10μg.On the contrary,at low dose of 0.1μg,the longer the incubation time of MSU,the more it slowed the activation of sodium current and reduced the peak sodium current amplitude.These indicated that the effect of MSU on the current of ND7/23 is time-dependent.After incubation with 10μg and 0.1μg MSU for ND7/23 20 h,the peak current was significantly different from that in the blank group.Compared with high dose,low dose more delayed the activation of sodium current and reduced the amplitude of peak sodium current.This indicated that MSU has a concentration-dependent effect on the sodium current of ND7/23 at the same time.2.3 Effect of MSU on Nav1.8 current in HEK293T CellsIncubation of HEK293T Cells with different doses of urate solution after different times,the current of Nav1.8 was recorded under voltage clamp.The results showed that after incubating HEK293T cells with 0.1μg MSU for 24 hours and 10μg MSU for 2hours respectively,the peak currents of activation and inactivation of Nav1.8 were both reduced.The steady-state activation and inactivation curves of Nav1.8 both moved toward depolarization.But there only steady-state inactivation showed a significant difference with the control.These indicated that the effect of MSU on delaying the activation of Nav1.8 current was not obvious and the inactivation is accelerated.2.4 Effect of TNC on sodium current in ND7/23 CellsAfter incubating ND7/23 cells with 0.5μg,1μg,1.5μg TNC for a certain period respectively,the sodium current was recorded under voltage clamp.The results showed that the sodium current amplitude could be significantly inhibited in each dose of TNC.These results indicated that different doses of TNC had different effects on ND7/23sodium current activation and inactivation,and were dose-dependent.2.5 Effect of TNR on sodium current in ND7/23 CellsAfter incubating ND7/23 cells with 0.5μg TNR for 2 and 24 h,the sodium current was recorded under voltage clamp.The results showed that the sodium current of TNR was significantly different from the blank group.This indicated that the activation and inactivation effects of TNR on sodium current in ND7/23 cells were different at different time periods,showed time dependence and an overall inhibition of sodium current amplitude.The longer the incubation time,the higher the degree of inhibition.2.6 Effect of TNC on Nav1.8 current in HEK293T cellsAfter incubating HEK293T cells with 0.2μg TNC for a certain period,the Nav1.8current was recorded under voltage clamp.The results showed that TNC increased the peak current of Nav1.8 significantly,and both steady-state activation and inactivation curves moved toward hyperpolarization.This indicated that TNC facilitates the activation of Nav1.8 current and delays its inactivation.2.7 Effect of TNR on Nav1.8 current in HEK293T cellsAfter incubating HEK293T cells with 0.2μg TNR for a certain period,the Nav1.8current was recorded under voltage clamp.The results showed that TNR increased the peak current of Nav1.8 significantly,the steady-state activation curve moved toward the hyperpolarization direction,and the steady-state inactivation curve shifted to the depolarization direction.These results indicated that the TNR accelerates the activation and inactivation of the Nav1.8 current.2.8 Effect ofβ4 subunit on Nav1.8 current in HEK293T cellsThe Nav1.8 current was recorded under voltage clamp after hNav1.8-GFP andβ4were transiently co-rotated for 24 h at a mass ratio of about 3:2.Results showed thatβ4inhibited the Nav1.8 current and made the steady-state activation curve shift to the depolarization direction.In conclusion,at the body level,MSU induces red,swollen and painful acute inflammation of gouty arthritis.In vitro,MSU inhibits the amplitude of Na~+current in the dose range of 0.1μg to 10μg.On the contrary,TNC alleviated the acute onset of MSU-induced gouty arthritis at the body level,increasing the magnitude of Nav1.8current at the ex vivo level.It is revealed that the acute occurrence of gouty arthritis induced by MSU may require the mediation and participation of Tenascins.It is further speculated that the pain caused by MSU is not only achieved by Na~+channel,and there may be more mechanisms involved.Therefore,the regulation of the occurrence of gouty arthritis from the perspective of Tenascins-Na~+channel can help to enrich the diagnosis and treatment strategy for the occurrence of this disease. |
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