| Objective:To study the expression of serum Thl/Th2 and the expression of Glial Fibrillary Acidic Protein(GFAP)and Brain-Derived Neurotrophic Factor(BDNF)in the dorsal raphe nucleus of the midbrain of rats with PCPA-induced insomnia,We would explore the mechanism of Hewei Anshen decoction in the treatment of primary insomnia.Methods:Thirty-two SPF-level Wistar male rats were randomly divided into blank group,model group,Hewei Anshen decoction group and diazepam group,with 8 rats in each group.A rat model of insomnia caused by PCPA was established by intraperitoneal injection of p-chlorophenylalanine(PCPA).Except for the blank group,the rats in the other groups were subjected to insomnia,once a day for 2 days.On the 3rd day of modeling,the rats in each group were given the corresponding method according to the body weight of 10 ml/kg,once a day for 7 consecutive days,and Chinese herbal decoction was given to the Hewei Anshen decoction group.The diazepam group was given a concentration of 92 mg/L diazepam aqueous solution,and both the blank group and the model group were given physiological saline.After 7 days of intragastric administration,rats in each group were intraperitoneally injected with 10%chloral hydrate(0.3 ml/100 g).The abdominal aorta was collected and centrifuged to obtain serum.The brain tissue was fixed on ice,and the specimens were stored at low temperature for testing.The levels of IFN-?,TNF-?,IL-4 and IL-10 in rat serum were detected by enzyme-linked immunosorbent assay(ELISA).The expression of GFAP and BDNF in the dorsal raphe nucleus was detected by immunohistochemistry.The images were observed and collected under light microscope.Image Pro Plus 6.0(IPP6.0)image processing software was used to determine the mean optical density(MOD)of glial fibrillary acidic protein(GFAP)and brain-derived neurotrophic factor(BDNF)positive reactants in the dorsal raphe nucleus.Result:1.Behavioral and weight changes in ratsAfter modeling by PCPA,it was observed that the hair of each group of rats in the model group was messy and shedding,the circadian rhythm disappeared,and the restlessness during the day was different from that of the blank group.Combined with the sodium pentobarbital sodium correction experiment,it suggested that the model was successful.After the corresponding method of gavage,compared with the body weight of the model group(230.50±8.35g),the body weight of the rats in the diazepam group(245.75±11.87g)and the Hewei Anshen decoction group(241.78±12.06g)increased significantly(P<0.01;P<0.05).2.Changes in serum Thl/Th2 levels in ratsCompared with the model group(32.846±8.247pg/ml),the IFN-? content in the Hewei Anshen decoction group(40.154±5.979pg/ml)was significantly increased(P<0.05).Compared with the model group(18.222±3.706pg/ml),the TNF-? content in the Hewei Anshen decoction group(9.802±2.908pg/ml)was significantly lower(P<0.01).Compared with the model group(46.921±5.386pg/ml),the IL-10 content in the Hewei Anshen decoction group(38.352±2.939pg/ml)was significantly lower(P<0.01).Compared with the model group(8.720±1.116pg/ml),IL-4 content in the Hewei Anshen decoction group(7.659±1.616 pg/ml)decreased(P>0.05);IFN-?/IL-4 ratio was significantly increased(3.794±0.890 vs 5.294±0.752,P<0.01).3.Changes of GFAP expression in the dorsal raphe nucleus of the midbrain of each groupThe GFAP-positive reactants in the dorsal raphe nucleus of each group were brown in the cytoplasm,the nucleus was light blue,and the cytoplasm was stained in a star shape with different lengths.As shown in the figure,the expression of GFAP positive products in the model group was less,and the expression of GFAP positive products in the blank group,diazepam group and Hewei Anshen decoction group was more.Compared with the blank group(0.0034±0.0010),the increase of GFAP MOD in the dorsal raphe nucleus of the rats of the Hewei Anshen decoction group(0.0035±0.0011)was not significant(P>0.05).Compared with the model group(0.0023±0.0014),the GFAP MOD of the dorsalraphe nucleus of the rats in the blank group(0.0034±0.0010),diazepam group(0.0033±0.0010)and Hewei Anshen decoction group(0.0035±0.0011)were significantly increased(P<0.01;P<0.05;P<0.01).4.Changes of BDNF expression in the dorsal raphe nucleus of the midbrain of each groupAfter immunohistochemistry,the BDNF positive reaction in the dorsal raphe nucleus of the rat midbrain was yellow or brown in the cytoplasm or nucleus.As shown in the figure,the expression of BDNF positive products in the blank group and the model group was less,and the expression of BDNF positive products in the diazepam group and the Hewei Anshen decoction group was more.Compared with the blank group(0.00848±0.00115),the BDNF MOD in the dorsal raphe nucleus of the rats in the diazepam group(0.00978±0.00124)and the Hewei Anshen decoction group(0.00955±0.00206)were significantly increased(P<0.01;P<0.05).Compared with the model group(0.00846±0.00184),the BDNF MOD in the dorsal raphe nucleus of the rats in the diazepam group(0.00978±0.00124)and the Hewei Anshen decoction group(0.00955±0.00206)were significantly increased(P<0.01;P<0.05).Conclusion:The mechanism of action of Hewei Anshen decoction on insomnia may be related to the shift the balance of Thl/Th2 toward the Thl direction by increasing the ratio of serum IFN-y/IL-4,increasing the expression of GFAP and BDNF in the dorsal raphe nucleus,which is related to the regulation of the body's immune system and central nervous system. |
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