| Background and aimsAlong with the advance of human civilization,more and more people pay more attention for the healthy problem.Non-communicable chronic diseases have become a threat to the people’s health all over the world.Diabetes(diabetes mellitus DM)causes a global public health problem and becomes more and more serious.It is estimated that in2010,the world has 34.5 million people died of the non-communicable diseases,from1990 to 2010,only during this periods,the DM mortality doubled,only in 2010,the people all over the word who died of DM increased to 1.3 million.Diabetic neuropathy is one of the most common chronic complications of DM,central nervous system and peripheral nerve can be affected.Painful diabetic neuropathy is the most severe complications which cost much money and have highest morbidity,some patients have significantly abnormal pain when under the mechanical stimulation,the common performance is burning pain,electric pain,tingling,allodynia and hyperalgesia,we called those mechanical pain-diabetic mechanical allodynia(DMA),DMA causes the problems such as anxiety,depression and sleep disturbance,seriously affect the patient’s quality of life.At present,the pathogenesis of DMA is still at the exploration stage,lack of effective treatment measures.Therefore,this research choose the islet beta cell damage of toxinsstreptozotocin,inject it on the healthy adult male SD rats,induced to type 1 DM rats model which we need,while DPN appears relatively late,through the subsequent behavior observation and mechanical stimulation experiment found that type 1 DM rats model can show the obvious abnormal pain behavior and at the same time have allodynia,together with the DM rat model induced by STZ have longer course of the disease and have significant mechanical pain,make it more suitable for research PDN.TRP channels arevery sensitive to temperature changes,the TRP channels among the tissues and in vitro studies confirmed that it can be used as mechanical sensitivity channels involved in mechanical stimulation,chemicals such as pain form,TRP channels has important role in the pathogenesis of DMA.This experiment adopts the methods of molecular biology and pharmacology behavior,some research showed that machinery sensitive TRP channels,such as TRPV1,TRPA1,TRPM8 channel’s changes of time and space expression in the spinal cord dorsal root ganglion,provide new way for effective clinical treatment.Methods(1)Rats were randomly divided into normal control group and experimental group,By intraperitoneal injection of STZ once time to establish type 1 DM rat model;(2)use von Frey filament to measure the mechanical paw retraction threshold(PWT)of DM rats model,observed the change of the PWT according to the experiment method,select the DMA rat model;(3)To filter out the DMA group rats model,were randomly divided into 1week,2 weeks,3 weeks,4 weeks group,according to the categories in the DMA rats model,take the DMA rat model’s DRG,save to-80℃ refrigerator;(4)using real-time fluorescent Quantitative PCR for detecting TRPV1、TRPA1、TRPM8 in the rat model’s DRG,observe the expression changes in each week group;(5)Using Western blot method for detecting TRPV1,TRPA1,TRPM8 in the rat model’s DRG,observe the expression changes in each week group.Results(1)The experimental results showed that the control SD rat’ s average blood glucose is 6.1-8.4 mmol/L;Three days after STZ injection,the male SD rat model ’s average blood glucose is 22.3 mmol/L,we can monitor a significant rise in blood sugar levels.1,2,3,4weeks later group of DM rats blood glucose fluctuation between 22.3 and 22.3 mmol/L,were significantly greater than 16.7 mmol/L,it means that we can successful preparation of DM rats model,compared with the control blood sugar levels,P < 0.01,the difference was statistically significant,along with the progression,part of the DM rat model’s blood sugar levels are very high,so the glucometer does not work.(2)DM rats for 3 days,1,2,3,4 weeks group weight fluctuations in 233.5 to 280.2 g,the weight compared with control group,P<0.01,the difference was statistically significant;the weight growth rate of DM rats model slowed significantly slower compared with the healthy rats who have adaptability of feeding,even some DM rat model’s weight go down.(3)7days after STZ injection,about 60% of DM rat models have DMA;Determination of mechanical pain threshold(PWT)in the control group on average 10 to15 g,3 days after STZ injection,compared with the normal control group,we found that the rat’s mechanical pain threshold is reduced in DM rats,a significant decrease in the seventh day,to the lowest in the 21 day,and maintain to 28 day,Compared with the control group’s PWT,P < 0.01,the difference was statistically significant.(4)The results showed that compared with the control group,the PCR results showed that in the DMA 7 day,TRPV1 mRNA expression is obviously increased,after a little lower but still at a high level;Using Western blotting method to detect the levels of protein,we found that in the DMA 7 days,the protein levels peaked,level and mRNA level changes after synchronization.TRPA1 mRNA and protein expression gradually over time increases,21 and 28 days highest levels.TRPM8 mRNA and protein expression level began to rise in a week,rose to the highest on DMA14 d and sustain at a high level.Conclusions(1)Through abdominal cavity to male SD rats one-off injection of STZ ways of successful preparation of DM rats model,the DM rat model can appear more drinks,food,DM associated symptoms such as urine,with von Frey filament to PWT level assessment,the study found that the DM rat model of 3 days,1,2,3,4 weeks group compared with the control of blood glucose,body weight,PWT,P < 0.01,the difference was statistically significant.Many related literatures suggest that using the DM rats which induced by STZ can not only have a mechanical pain,but also have thermal hyperalgesia and cold pain through the hot plate and cold plate experimental.(2)In DRG of DMA model rats,the expression of TRPA1,TRPV1,TRPM8 showed a transient trend of increase at varied time point,TRPV1 and TRPM8 appeared rising trend in the early days,TRPA1 appeared rising trend in the late days.Different TRP channels do not ride at the same time,indicated that the mechanism of DMA is a complicated process,three different TRP channels are involved in the development of the DMA. |
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