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Objective:To evaluate the role of transient receptor potential melastatin 2 (TRPM2)-calcineurin A (CnA)-dynamin-related protein 1 (Drp1) pathway in propofol-induced reduction of renal injury induced by hepatic ischemia-reperfusion (I/R) in mice.Methods:Twenty-four SPF male C57BL6 mice, aged 8 weeks, weighing 20-23 g, were divided into 4 groups ( n=6 each) using a random number table method: sham operation group (group S), hepatic I/R group (group IR), propofol group (group P) and TRPM2 agonist (ADPR) combined with propofol group (AP group). Hepatic I/R injury was induced by occluding the portal vein and hepatic artery supplying the left and middle lobes of the liver for 60 min followed by reperfusion in anesthetized rats.In group P, 0.2 ml normal saline was injected intraperitoneally at 1 h before establishing the model and 1% propofol 30 mg/kg was injected intraperitoneally at 30 min before establishing the model.In group AP, ADPR 10 mg/kg (in 0.2 ml of normal saline) was injected intraperitoneally at 1 h before establishing the model, and 1% propofol 30 mg/kg was injected intraperitoneally at 30 min before establishing the model.The equal volume of normal saline was given intraperitoneally at 1 h and at 30 min before establishing the model in group S and group IR.Blood samples were taken from the eyeballs for determination of the levels of serum urea nitrogen (BUN), creatinine (Cr), aminotransferase (ALT) and aspartate aminotransferase (AST) at 6 h of reperfusion.The animals were then sacrificed and the kidney tissues were taken, the ultrastructure of myocardial mitochondria was observed using transmission electron microscopy, the average diameter of mitochondria was calculated, and the expression of TRPM2, CnA, phospho-Drp1 Ser637 (p-Drp1 Ser637) and cleaved caspase-3 was detected (by Western blot). Results:Compared with group S, the concentrations of serum BUN and Cr were significantly increased, the expression of TRPM2, CnA and cleaved caspase-3 in kidney tissues was up-regulated, the expression of p-Drp1 ser637 was down-regulated, and the average diameter of mitochondria was shortened in IR, P and AP groups ( P<0.05). Compared with group IR, the concentrations of serum BUN and Cr were significantly decreased, the expression of TRPM2, CnA and cleaved caspase-3 in kidney tissues was down-regulated, the expression of p-Drp1 Ser637 was up-regulated, the average diameter of mitochondria was prolonged ( P<0.05), mitochondrial injury was attenuated, and no significant change was found in the serum ALT and AST concentrations in group P, and no significant change was found in concentrations of BUN and Cr in serum in group AP ( P>0.05). Compared with group P, concentrations of BUN and Cr in serum was significantly increased, the expression of TRPM2, CnA and cleaved caspase-3 in kidney tissues was up-regulated, the expression of p-Drp1 Ser637 in kidney tissues was down-regulated, and the average diameter of mitochondria was shortened ( P<0.05), and mitochondrial injury was accentuated in group AP. Conclusion:The mechanism of propofol-induced reduction of renal injury induced by hepatic I/R is related to inhibiting the expression of TRPM2 in kidney tissues, decreasing the level of intracellular CnA and inhibiting dephosphorylation of Drp1 Ser637 in mice.
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Objective To evaluate the relationship between cold hyperalgesia and trafficking of transient receptor potential melastatin 8 (TRPM8) to cell membrane in the dorsal root ganglion (DRG) of rats with neuropathic pain (NP).Methods Ninety-six healthy male Sprague-Dawley rats,aged 10-12 weeks,weighing 250-280 g,were divided into sham operation group (S group,n=48) and NP group (n =48) using a random number table.NP was produced by chronic constriction injury to the sciatic nerve.The number of paw lifts on the cold plate and mechanical paw withdrawal threshold (MWT) were measured on 1 day before operation and 1,4,7,10 and 14 days after operation.Rats were sacrificed after behavioral testing,and ipsilateral DRGs of the lumbar segment (L46) were dissected tor detection of the expression of TRPM8 in total and membrane proteins by Western blot,and the ratio of TRPM8 expression in the membrane protein to that in the total protein (m/t ratio) was calculated.Results Compared with group S,the number of paw lifts on the cold plate was significantly increased,the MWT was decreased,the expression of TRPM8 in total and membrane proteins was up-regulated,and m/t ratio was increased on postoperative days 4,7,10 and 14 in group NP (P＜0.05 or 0.01).In group NP,the number of paw lifts on the cold plate was gradually increased with the prolongation of time after operation and reached the peak on postoperative day 10,maintaining at the peak until postoperative day 14;the MWT was gradually decreased and reached the lowest level on postoperative day 10,maintaining at the lowest level until postoperative day 14;the expression of TRPM8 in total and membrane proteins and m/t ratio were gradually increased with the prolongation of time after operation and reached the peak on postoperative day 10,maintaining at the peak level until postoperative day 14 (P＜0.01).Conclusion The mechanism underlying the development of cold hyperalgesia is related to enhanced trafficking of TRPM8 to cell membrane in DRGs of rats with NP.
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Objective To evaluate the role of glial cell line-derived neurotrophic factor family re-ceptor alpha3(GFRα3)in the expression and membrane trafficking of transient receptor potential melasta-tin 8(TRPM8)in the dorsal root ganglion(DRG)during cold hyperalgesia in rats with neuropathic pain (NP). Methods Thirty-two healthy adult male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, in which intrathecal catheters were successfully implanted, were divided into 4 groups(n=8 each) using a random number table: sham operation plus GFRα3 dsRNA group(Sham+dsRNA group), sham operation plus GFRα3 siRNA group(group Sham+siRNA), NP plus GFRα3 dsRNA group(group NP+dsRNA)and NP plus GFRα3 siRNA group(group NP+siRNA). NP was produced by chronic constriction injury to the sciatic nerve. At 10-30 days after operation, GFRα3 dsRNA 10 μg∕20 μl was intrathecally injected once a day for 4 consecutive days in Sham+dsRNA and NP+dsRNA groups, and 10 μg∕20 μl GFRα3 siRNA, of which the sense strand was modified with 2′-O-methyl and 5′-cholesterol, was intrathe-cally injected once a day for 4 consecutive days in Sham+siRNA and NP+siRNA groups. The number of paw lifts on the cold plate, mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency (TWL)were measured on 1 day before operation and 10, 11, 12, 13(before intrathecal injection)and 14 days after operation. The rats were sacrificed after the last behavioral testing, and ipsilateral DRGs of the lumbar segment(L4-6)were dissected for detection of the expression of GFRα3 and TRPM8 in total and membrane proteins by Western blot, and the ratio of TRPM8 expression in the membrane protein to that in the total protein(m∕t ratio)was calculated. Results Compared with group Sham+dsRNA, the number of paw lifts on the cold plate was significantly increased, the MWT was decreased, and TWL was shortened after operation in NP+dsRNA and NP+siRNA groups, the expression of GFRα3 and TRPM8 in total and membrane proteins was significantly up-regulated, and m∕t ratio was increased in group NP+dsRNA, and the expression of GFRα3 in DRGs was significantly down-regulated(P<001), and no significant change was found in the expression of TRPM8 in total and membrane proteins or m∕t ratio in group NP+siRNA(P>005). Compared with group NP+dsRNA, the number of paw lifts on the cold plate was significantly de-creased, the expression of GFRα3 and TRPM8 in total and membrane proteins was down-regulated, m∕t ra-tio was decreased(P<001), and no significant change was found in MWT or TWL in group NP+siRNA (P>005). Conclusion GFRα3 in DRGs can up-regulate the expression of TRPM8 and enhance the membrane trafficking of TRPM8, which may be involved in the maintenance mechanism of cold hyperalgesia in rats with NP.
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Objective: To investigate the changes of TRPM8 expression in dorsal root ganglion(DRG) in the rat model of chronic constriction injury(CCI) of the sciatic nerve. Methods: Seventy-two male SD rats weighing 250~280 g were randomly divided into 2 groups (n = 36 each): group I (CCI) and group II (sham operation). The threshold of cold hyperalgesia, heat hyperalgesia and mechanical hyperalgesia were measured before operation (baseline) and at 1, 4, 7, 10 and 14 d after operation. Six rats were killed at each time point in each group. The L5 DRGs ipsilateral to nerve injury were dissected out for determination of transient receptor potential melastatin 8(TRPM8) by immunohistochemical assay. Results: The thresholds of cold, thermal and mechanical stimuli started to decrease at 4 d after CCI in operation group and maintained at a relatively low level until the end of experiment. The cold and thermal hyperalgesia peaked at 10 d after operation and mechanical hyperalgesia at 14 d. Immunohistochemical assay demonstrated that expression of TRPM8 were increased in L5DRG on the operated side significantly at 4 d after CCI and reached the peak at 10 d and was maintained at a high level until the end of experiment. Conclusion: The upregulation of TRPM8 in DRG involved in the mechanism of neuropathic pain.
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Objective To investigate the effects of acidosis on the current change of transient receptor potential channel M7 (TRPM7) and collagen production in mouse cardiac fibroblast (MCFs), and to explore the pathophysiological function of TRPM7 on the cardiac fibrosis. Methods (1) The model of MCFs was established and isolated. (2) MCFs was subcuhured. (3) Patch clamp technique was used to observe the current characteristics of TRPM7 in low PH solutions. (4) The influence of acidic condition on Ca2+ influx in MCFs was recorded by calcium fluorescent indicators. Results (1) There was a high level expression of TRPM7 in MCFs and the electrophysiological characteristics of TRPM7-like (TRPM7L) was similar to that of TRPM7. (2) Ca2+ influx was increased in acidic condition, and the F340/F380 ratio was increased from 1.0 to 4. 6 at pH 4.0 compared with pH 7.0 (t=2.72, P<0.01). Conclusions (1) TRPM7 is the molecular basis of the native TRPM7L in MCFs and TRPM7L plays an important role in Ca2+ influx. (2) The pathophysiological function of MCFs is influenced by regulation of Ca2+ influx mediated by TRPM7L in the condition of acidosis.
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Objective To investigate the changes of TRPM 7-like current in mouse cardiac fibroblast(CFs) after myocardial infarction and the effect of myocardial ischemia on the TRPM 7 expression and current. Methods (1) The model of myocardial infarction was made and CFs were isolated;(2) CFs were cultured and infected by TRPM 7 siRNA;(3) The effects of myocardial ischemia on TRPM 7 current were recorded by whole cell patch clamp technique;(4) The influences of myocardial isehemia on Ca2+ influx in CFs were recorded by Ca2+ fluorescence imaging. (5) The effects of ischemia on total collagen content of CFs were studied. Results (1) Ca2+ inward current of CFs was increased after myocardial infarction [(16.2±1.7) vs. (7.4±0.7) pA/pF, P<0.053];(2) TRPM 7 current was reduced by 90% after siRNA infection;(3) The total collagen content of CFs after ischemia was approximately 2.3-fold higher than basic value. Conclusions Ca2+ influx in CFs plays an important role in the pathophysiological process of myocardial fibrosis.