The Effect Of α7nAChR On Crush Syndrome

Backgrounds and ObjectivesCrush injury is defined as compression of the extremities or other parts of the body,resulting in muscle swelling and/or neurological disturbances in the affected areas of the body.Based on crush injury,CS is characterized by systemic symptoms due to rhabdomyolysis including acute kidney injury,hypovolemic shock,and metabolic disorders（e.g.,hyperkalemia）,which appears to be common in disasters,wars,and acts of terrorism.CS in violent earthquakes,with an incidence about 2¹5%,is the most frequent cause of death,apart from trauma.Up to 20% crush victims died of cardiac arrest caused by hyperkalemia or hypovolemic shock in a short time.However,safe and effective drugs to reduce on-site mortality in CS are clinically vacant,due to lack of definite diagnosis.Anisodamine（Ani）is a belladonna alkaloid isolated from the Chinese medicinal herb Scopolia tangutica Maxim of the Solanaceae family indigenous to Tibet.It is used clinically to improve blood flow in circulatory disorders such as septic shock and disseminated intravascular coagulation.Our previous studies found that activation of α7nicotinic acetylcholine（ACh）receptor（α7nAChR）is involved in the antishock effect of Ani.As known,insulin can promote entrance of potassium into cells.The effect of insulin on serum potassium has been attributed to activation of Na⁺/K⁺-ATPase.Chronic exposure to nicotine could enhance insulin sensitivity via α7nAChR.We speculated that activation of α7nAChR with Ani could decrease serum potassium through elevation of insulin sensitivity.Moreover,hyperkalemia is reported to be mainly found in adult male victims in CS after earthquake and E₂ was documented to decrease the plasma K⁺concentration.E₂ was reported to stimulate activity and expression of Na⁺/K⁺-ATPase in cell and vascular smooth muscle cell by mechanism that involved the participation of insulin receptor substrate-1/phosphatidylinositol-3 kinase/protein kinase B signaling in rats.While,the relation among E₂,hyperkalemia,and insulin sensitivity remains unclear in CS.The present work was designed to test the effec of α7nAChR activation on reduction of on-site mortality in CS,and the role of E₂ in α7nAChR activation-mediated decline in on-site mortality of mice with CS.Experimental protocolsExperiment 1: Effect of Ani on Mortality and Serum Biochemicals in Rats with CS.Rats were randomly divided into four groups:（1）normal（2）control（3）Ani-pre: rats received received Ani at 30 min before decompression;（4）Ani-post: rats received Ani at 1h after decompression.CS models were established in group 2-4.Survival time was monitored for 24 h after decompression.CS models were established in another five groups of rats as listed above.Blood samples of all the rats were collected at 6 h after decompression,and serum CK,CK-MB,BUN,Scr,K⁺,Na⁺,and Cl-levels were measured.Experiment 2: Effect of Ani on Insulin Sensitivity in Rats with CS.CS models were established as listed in Experiment 1.Serum insulin and glucose levels were measured at 6 h after decompressionExperiment 3: The Role of α7nAChR In the Effect of Ani on Mortality,Serum K⁺ and Insulin Sensitivity in C57BL/6 Mice with CS.C57BL/6 mice were randomly divided into five groups:（1）control（2）Ani（3）MLA（4）Ani+ MLA（5）PNU.CS models were established.Ani or PNU was given at 30 min before decompression,and MLA was given 30 min earlier.Survival time was monitored for 24 h after decompression.CS models were established in another five groups of C57BL/6 mice as listed above.Blood samples were collected at 6 h after decompression,and serum K⁺,insulin,and glucose levels were measured,as well as in a “normal” group.Experiment 4: Role of α7nAChR In the Effect of Ani on Mortality,Serum K⁺ and Insulin Sensitivity in α7nAChR Knockout Mice with CS.Normal saline or Ani was given to α7-/-mice and wild-type controls at 30 min before decompression.Survival time was monitored for 24 h after decompression,and serum K⁺,insulin,and glucose levels were measured at 6 h after decompression.Experiment 5: Effect of Ani on mortality and serum K⁺ in rats with hyperkalemia.Rats were randomly divided into two groups,receiving normal saline or Ani,and KCl Experimental protocolswas given 30 min later.Blood samples were collected at 30 min after KCl injection and from heart for those just died,and serum K⁺ level was measured.Survival time was monitored for 24 h after KCl injection in another two groups.Experiment 6: Involvement of α7nAChR in the effect of Ani on extracellular K⁺in vitro.Myotubes were pretreated for 30 min with Ani,ACh,Ani + ACh,nicotine,or PNU prior to exposure to KCl,and extracellular K⁺ and glucose levels were measured 6 h later.In another set of experiments,myotubes were pretreated for 1 h with mecamylamine,MLA,or hexamethonium,and then for 30 min with Ani + ACh or nicotine prior to exposure to KCl,and extracellular and glucose levels were measured 6 h later.Experiment 7: Insulin signaling pathway mediated the effect of Ani on extracellular K⁺ in vitro.In these experiments,myotubes were divided into five groups: 1)normal;2)control;3)Ani + ACh;4)HNMPA-（AM）3;5)Ani + ACh + HNMPA-（AM）3.Myotubes were pretreated for 1 h with HNMPA-（AM）3,and then for 30 min with Ani + ACh prior to exposure to KCl.Phosphorylation of Na⁺/K⁺-ATPase was then detected with immunofluorescence staining and confocal microscopy.Expression of α7nAChR and insulin receptor was also examined in normal C2C12-differentiated myotubes with immunofluorescence.Myotubes were pretreated for 1 h with HNMPA-（AM）3,LY 294002,rapamycin,stattic,or ouabain,and then for 30 min with Ani + ACh prior to exposure to KCl,and extracellular K⁺ and glucose levels were measured 6 h later.Experiment 8: Gender Difference of Serum K⁺,E₂,Insulin Sensitivity,and Mortality in Mice with CS.Female mice were randomly divided into normal group and CS model group（mice received compression）.Twelve male mice were divided and treated similarly.Blood samples were collected at 6 h after decompression,and serum K⁺,E₂,insulin,and glucose levels were measured,as well as in “normal” group.CS models were established in another group of mice.Survival time was monitored for 24 h after decompression.Experiment 9: Influence of Activation of α7nAChR on Serum K⁺and E₂ in Mice with CS.Male mice were randomly divided into five groups: 1)normal: mice received normal saline;2)CS model: mice received normal saline;3)MLA;4)Ani);5)Ani + MLA.CS models were established in group 2-5.Ani was given i.p.at 30 min before decompression,and MLA was given 30 min earlier.Blood samples were collected at 6 h after decompression,and serum K⁺ and E₂,levels were measured,as well as in “normal” group.Experiment 10: Influence of OVX on Serum K⁺,E₂,and Insulin Sensitivity in Mice with CS.Female mice were randomly divided into four groups 2 weeks after OVX surgery: 1)normal: mice received normal saline;2)CS model: mice received normal saline;3)Ani;4)E₂.CS models were established in the later three groups.Four groups of female mice were treated similarly 2 weeks after sham operation for OVX surgery.Ani and E₂ were given at30 min before decompression.Blood samples were collected at 6 h after decompression,and serum K⁺,E₂,insulin,and glucose levels were measured,as well as in “normal” group.Experiment 11: Effects of E₂ on Mortality in Mice with CS.Female mice were randomly divided into three groups 2 weeks after OVX surgery: 1)CS model: mice received normal saline;2)Ani;3)E₂.CS models were established in all the groups.Three groups of female mice were treated similarly 2 weeks after sham operation for OVX surgery.Ani and E₂ were given at 30 min before decompression.Survival time was monitored for 24 h after decompression.Experiment 12: Influences of Activation of α7nAChR on Blood Pressure in Rats with CS.Male rats were randomly divided into five groups: 1)normal: rats received normal saline;2)CS model: rats received normal saline;3)MLA;4)Ani;5)Ani + MLA.After catheterization and recovery for 2 d,CS models were established in group 2-5.Ani was injected at 30 min before decompression,and MLA was injected 30 min earlier.Blood pressure was monitored for 3.5 h since 30 min before decompression,as well as in“normal” group.ResultsExperiment 1Compared with control group,Ani administration at 30 min before decompression or at 1 h after decompression could significantly decrease the on-site mortality and significantly decrease serum K⁺,Scr,CK,CK-MB and urea nitrogen in CS.Experiment 2Compared with control group,Ani administration at 30 min before decompression or at 1 h after decompression could significantly improve insulin sensitivity in CS.Experiment 3MLA attenuated the effect of Ani on on-site mortality in CS.PNU could significantly decrease the on-site mortality and improve the insulin sensitivity in CS.Experiment 4Ani at 30 min before decompression significantly decreased the mortality rate,prolonged survival time,and decreased serum K⁺ level in WT mice,but had no effect inα7nAChR mice.Experiment 5Ani at 30 min before KCl injection could decrease the mortality rate of rats with hyperkalemia by decreasing the serum K⁺.Experiment 6Both Ani and Ani + ACh decrease extracellular K⁺and glucose.Ani + ACh have better effect than single Ani administration.Mec and MLA could attenuate the effect of Ani+ ACh or Ani.Nicotine and PNU decrease extracellular K⁺ and glucose.Experiment 7Ani + ACh significantly increased phosphorylation of Na⁺/K⁺-ATPase in cultured myotubes.Pretreatment with HNMPA-（AM）3 attenuated the effect of Ani and ACh on Na⁺/K⁺-ATPase phosphorylation to a level that was significantly lower than control group.Extracellular K⁺ and glucose in cultured myotubes were significantly decreased by Ani and ACh.Pretreatment with HNMPA-（AM）3 or ouabain attenuated the effect of Ani and ACh on extracellular K⁺to a level that was significantly higher than control group.Pretreatment with LY 294002,rapamycin,or stattic also attenuated the effect of Ani and ACh on extracellular K⁺.Experiment 8There is no significant difference in on-site mortality between female mice and male mice with CS.Female mice with CS have higher insulin sensitivity,higher serum E₂ level and lower serum K⁺ than adult male mice with CS.Experiment 9The activation of α7nAChR could decrease serum K⁺and improved serum E₂ level in mice with CS.Experiment 10OVX improved the serum K⁺,and decreased insulin sensitivity and serum E₂ level of mice with CS.Experiment 11E₂ decreased the on-site mortality of mice with CS.Experiment 12SBP,DBP and MBP decreased gradually post-decompression in rats with CS compared with normal group.Ani treatment group had higher SBP,DBP and MBP after 2.5h post-decompression compared with control group.MLA alleviated the effect of the Ani on the blood pressure.Each group had no significant difference in HR.ConclusionActivation of α7nAChR with Ani decreases on-site mortality in CS;such effect of activating α7nAChR is partially due to decline of serum potassium and partly due to theimprovement of blood pressure;Ani decreases serum potassium through insulin signaling-Na⁺/K⁺-ATPase pathway.Moreover,adult male rat tends to suffer from hyperkalemia after decompression and this could be contributed to the effect of E₂ on the enhancement of insulin sensitivity.