Role Of Nrg1-erbB Signaling In Morphine Tolerance In The Mice Spinal Cord

Objective To investigate the role of Nrg1-erb B in the development of morphine tolerance in mice.Methods The experiment consists of two parts,grouped as follows: Part I: Eighteen Male KM mice were randomly divided into 2 groups(n=9):morphine + saline group(MT group)and normal saline group(NS group).MT group was given subcutaneous injection of morphine(10mg / kg),NS group was given subcutaneous injection of saline.Treatment was given twice a day for seven consecutive days.At the 1st,3rd,5th and 7th day,the latency of tail flick was measured by the Tail flick test,and the possible maximum analgesic effect(% MPE)was calculated.After 7 days of morphine administration,three mice of each group were sacrificed to take the lumbar enlargements of spinal cords which were used to investigate the protein expression of P-erb B2 by Western blot and the m RNA level of Nrg1 detected by real-time PCR.Therest of the mice were used for immunofluorescence staining for Iba1,a specific marker for microglia.Part II: Thirty male KM mice were randomly divided into 5 groups(n=6): vehicle + MT group(V + MT group),lapatinib group(L group),saline group(SN group),MT group and lapatinib + morphine group(L +MT group).L group was intraperitoneal injected with 300 μl(1 μg / μL)lapatinib two hours before 120 μL of normal saline was given subcutaneously as control group of morphine.V+MT group was intraperitoneal injected with 300μL vehicle(8%DMSO+5%Tween-20+Saline),followed by subcutaneously injection of 120μL(2.5μg / μL)morphine 2 hours later.L+MT group was administrated with 300μL lapatinib,followed by subcutaneously injection of 120μL(2.5μg / μL)morphine 2 hours later.The drugs were injected twice a day for seven consecutive days.On day1,3,5,7,the tail flick test was conducted thirty minutes after the drug injection in the morning.On day7 after conducting tail flick test,3 mice in each group were sacrificed to take the lumbar enlargements in spinal cords which were used to investigate the protein activation of erbb2 by Western Blot.The rest of the mice were intracardialy perfused with 4% ice-cold paraformaldehyde(PFA)in 0.1 M phosphate buffer saline(PBS,PH=7.4)to remove the lumbar enlargements which were utilized to investigate the activation of microglia by immunofluorescence.Results 1.The MPE% in the MT group was the highest in the subcutaneous injection of morphine at the first day,and the analgesic effect of morphine was the strongest.On the third day,the MPE% of MT group decreased,and the analgesic effect of morphine was significantly decreased(P <0.001)on day 7 of morphine administration.Indicating that the morphine tolerance model was successfully established.Real time-PCR result showed that the m RNA level of Nrg1 in the spinal cord of the MT group was significantly higher than that of the NS group(P <0.05).The result of Western Blot indicated that the expression of P-erbb2 protein in the spinal cord was significantly higher than that in the SN group and MT group(P <0.05).The expression of Nrg1-erb B signaling was up-regulated by the development of morphine tolerance.The results of immunofluorescence and positive cell counting showed that compared with SN group,the morphology of microglia in spinal dorsal horn of MT group was changed and the positive cell number was significantly increased(P <0.001).2.Mice were intraperitoneal injected with lapatinib,a specific erb B2 receptor blocker,two hours before morphine administration.The MPE% of L + MT group,MT group and V + MT group reached the highest value on the first day of administration,and there was no significant difference(P> 0.05).The extent of the decreased MPE% of L + MT group was lower than that of the latter two groups(P<0.001).There was no significant difference in MPE% between MT group and V + MT group(P> 0.05),and no significant difference in MPE% between SN group and L group(P> 0.05).Immunofluorescence results showed that the positive cell number of microglia in spinal dorsal horn of L + MT group was significantly lower than that of MT group and V + MT group(P<0.001).There was no significant difference in the number of positive cells between L + MT group and SN group and L group(P> 0.05).Indicating that lapatinib can inhibit the activation of microglia.Western Blot results showed that the expression of P-erbb2 protein in L + MT group was lower than that in MT group and V + MT group(P<0.05).Compared with L group and SN group,the expression of P-erbb2 protein in MT group and V +MT group was significantly increased(P<0.001).Conclusion In general,the Nrg1-erb B signaling is involved in the development of morphine tolerance,and its mechanism may be related to Nrg1’s effect to microglia activation.