The Effects Of Estrogen On The Gene Expressions Of Substance P And Its Receptor In The Midbrain Of Rat Migraine Model

[Objective] Migraine is a common neurological disorder, the exact pathogenesis is still unclear. A variety of neuropeptides such as substance P (SP), calcitonin gene-related peptide, neurokinin A have been implicated in the occurrence of migraine. SP, as one of the most important neurotransmitters, might play an important role in sensory information transmission and pain modulation. Currently, it has been thought SP might play a dual role on the transmission of pain messages:on the one hand, SP, as an afferent neurotransmitters of injury information, might exert promotion/facilitation effects in the first synapse of pain sense transmission in the spinal dorsal horn and spinal trigeminal nucleus; on the other hand, SP, with modulating pain sense and reducing susceptibility to pain, might exhibit an analgesic effect in brain. Midbrain is of crucial significance in pain modulation, studies addressing migraine showed that numerous SP-positive and SP receptor-positive pericaryons and fibers are observed in periaqueductal gray and rapheal nuclei. However, no quantitative study reported SP mRNA in midbrain. The incidence of migraine in women is higher than in man. It has reported part of women experienced migraines only during the premenstrual or menstrual phase of their cycles, suggesting that lower estrogen levels can cause migraine. Research has shown estrogen supplements can control and prevent the menstrual migraine occurrence, but whether the mechanism is associated with SP and its receptor in brain is not clear. By using Wistar rats to induce migraine model by exogenous estrogen administration to ovariectomized rats with a low estrogen level, the present study investigated the changes of expressions of SP and its receptor in the brain to different dose of estrogen in migraine rat. Therefore, the present study adopts real-time quantitative PCR technique, which is considered as the most accurate, most repeatable and generally accepted detection method for nucleic acid molecular from a view of quantitation and qualitation, to conduct an absolute quantitative study addressing SP mRNA in midbrain of migraine rats, thus to discuss the changes of SP and its receptor gene during the pathophysiology by the different doses of estrogen in rat migraine model.[Methods] 1.Establishing quantitative standard curves of SP mRNA and NK1 mRNA by extracting total RNA from midbrain of a rat through real time PCR.2. Grouping, model establishment and interventions:Totally 36 healthy adult Wistar rats were ovariectomized, then 7 days of feeding later, they were divided into six groups at random, namely control group, migraine group, low-and high-dose of estrogen treated groups, low-and high-dose of estrogen control groups (n=6).2 mL/kg peanut oil was injected into rat cervical and back in the control group, low-and high-dose of estrogen control groups. Experimental migraine models were established through subcutaneous injection of 10 mg/kg nitroglycerin into rats in the migraine group, low-and high-dose of estrogen treated groups. The models were considered success upon the appearance of head complaint such as two ears reddish, frequently scratching head with forepaws, increasing number of climbing cage, biting tails, to and fro movement, etc.2. Detect SP and its receptor mRNA expression with real-time PCR:Two hours following nitroglycerin injection, rats were anesthetized with 10% chloral hydrate (0.3 mL/100 g) and then decapitated. The midbrains were isolated and immediately placed in liquid nitrogen, preserved in a refrigerator at-70℃.50-100 mg of midbrain tissues was used to extract the total RNA followed the protocol. Subsequent to gel electrophoresis, total RNA quality and concentration were determined with ultraviolet spectrophotometer. The ratio of A260/A280 should maintain at 1.9-2.0, RNA concentration was calculated according to the formula:RNA concentration (μg/μL)= A260×attenuation multiple×40/1000.20μL reaction system comprised RNA 300 ng with reverse transcriptase. The synthetized cDNA was preserved at-70℃for later use. According to SP and NK1 receptor specific primer, the SP and NK1 receptor genes were synthetized with PCR apparatus. PCR amplification product was processed into 2% agarose gel electrophoresis, cutting target band, and recovered. The purified target gene was conjugated with pMD-18T carrier and transformed into E.coli DH5a competent cell. Subsequent to Ampicillin screening, plasmid extraction commenced. followed by restriction endonuclease HindⅢand BamHⅠdouble digestion, sequencing and identification. The absorbance value of the extracted plasmid at 260 nm was measured, copy number was also calculated. Following sterile water 10-fold serial dilution and subpackage, the standard specimen was preserved at -20℃. Different concentrations of plasmid standard specimens and synthetized cDNA was processed into quantitative PCR. The SP and NK1 receptor mRNA content in rat midbrain was calculated based on the melting curve.SP mRNA copy number was expressed as mean±SD and analysis of variance was applied for comparison among groups. The level of significance was set to p< 0.05[Results] 1.By using the method of real-time quantitative PCR, we successfully established standard curve of SP gene and NK1 receptor gene.2. The succes rate for inducing migraine model is 100%.3. The SP and its receptor mRNA expression levels:SP and NK1 receptor mRNA levels in rat midbrain was calculated according to the standard plot. Statistical analysis showed that, the migraine group exhibited an obviously lower level of SP mRNA expression than control group (P< 0.05); compared with control group, migraine group, low-and high-dose of estrogen treated groups, the SP mRNA expression was enhanced in low-and high-dose of estrogen control groups (P< 0.05). No significant differences were found with SP mRNA expression among high-dose of estrogen treated group and migraine group (P> 0.05). There was no significance found with NK1 receptor mRNA expression between migraine group and control group (P> 0.05). Compared with control group, migraine group, low-dose of estrogen treated group, low-and high-dose of estrogen control groups, the SP mRNA expression was enhanced in high-dose of estrogen treated groups (P< 0.05).[Conclusion] The present study has successfully detected the expressions of SP mRNA and the NK1 mRNA gene by establishing the standard curve through using the real-time quantitative PCR, and examined the effect of different dose of estrogen on the expressions of SP mRNA and NK1 mRNA in the course of migraine. The results showed the level of SP mRNA has been down-regulation in the midddle brain of the migraine rats treated by nitroglycerin. Estrogen can increase the expression of SP mRNA in the rat without headache, but cannot effect the expressions of SP mRNA and NK1 mRNA in nitroglycerin induced migraine rat.