Mechanism Of Biorhythm Disturbance In Perimenopausal Liver Stagnation Pattern Rats From Neuroendocrine Changes

Objective1.Serum metabolomics and hippocampal proteomics were used to screen serum biological markers of perimenopausal liver stagnation pattern,analyse key regulatory signalling pathways in perimenopausal liver stagnation pattern,and biorhythm gene expression changes were examined to investigate the biological basis of perimenopausal liver stagnation pattern.2.From the perspective of correspondence between prescriptions and pattern,Chaihu Shugan San(CSS)was used to verify the mechanism of action of biorhythm disorders in perimenopausal liver stagnation pattern.Methods1.50 8-week-old SD female rats were selected and randomly divided into 20 in the sham-operated group and 30 in the modeling group.The modeling group underwent bilateral ovarian removal surgery to establish a perimenopausal model,and then randomly divided into perimenopausal(PP)group(10),perimenopausal liver stagnation(PLS)group(10),and CSS group(10).The sham-operated group only removed the adipose tissue next to the ovaries,and then randomly divided into the normal group(10)and liver stagnation pattern(LS)group(10).LS group,PLS group and CSS group were subjected to chronic mild unpredictable stimulation to establish liver stagnation pattern model.The rats in the CSS group were gavaged for 21 days at a dose of 3 g/kg/d,and an equal volume of 0.9% Na Cl solution to the rest of the rats.2.Vaginal exfoliated cell smears and enzyme-linked immunosorbent assays(ELISA)were used to measure serum levels of estradiol(E2),follicle-stimulating hormone(FSH),and luteinizing hormone(LH)in rats with the aim of demonstrate the PP model.Behavioural experiments and body weight were used to demonstrate the LP model.3.Serum levels of 42 neurotransmitters in rats were examined by metabolomics.4.Hippocampal protein levels were examined by proteomics and bioinformatics was used to analyse differential signalling pathways.5.m RNA expression of CLOCK,BMAL1,PER1 and PER2 in rat hippocampus were measured by q-PCR.6.Protein levels of CLOCK,BMAL1,PER1 and PER2 in rat hippocampus were determined by Western blot.Results1.Microscopic observation showed that the cells shed from the vagina of rats after removal of ovaries were dominated by a large number of leukocytes.2.Behavioral experiments: Compared with the normal group,the total distance moved,central area dwell time,vertical times and sucrose water consumption rate decreased in LS group and PLS group(P<0.05,P<0.01),and the swimming immobility time increased in LS group(P<0.05).Compared with PLS group,the number of verticals increased in PP group and CSS group(P<0.05,P<0.01),and the sucrose water consumption rate increased significantly in CSS group(P<0.01).Body weight: compared with the normal group,the body weight and the growth rate in LS group and PLS group decreased(P<0.01).Compared with PLS group,the body weight and growth rate increased in PP group and CSS group(P<0.01).E2,FSH and LH : compared with the normal group,the E2,FSH and LH decreased in PP group and PLS group(P<0.01),and the FSH and LH decreased in LS group.Compared with PLS group,the FSH increased in CSS group(P<0.05).3.Neurotransmitter:Compared with the normal group,5-hydroxyindoleacetic acid,homovanillic acid and spermine decreased(P<0.05)in LS group,5-serotonin and acetylcholine increased(P<0.05)in LS group;acetylcholine and melatonin increased(P<0.05)and homo-vanillic acid and spermine levels decreased(P<0.05)in PLS group.Compared with PLS group,5-hydroxyindoleacetic acid and glutamine levels increased in PP group(P<0.05),5-serotonin increased in LS group(P<0.05),and acetylcholine and melatonin decreased significantly(P<0.01),while 5-hydroxy-indoleacetic acid,glutamine and hypericin increased in CSS group(P<0.05).Acetylcholine and melatonin decreased in CSS group(P<0.05).4.Proteome quantification: In the protein GO function enrichment analysis,the functions related to neuroendocrine were significantly enriched in the 2 comparison groups.In the differential protein KEGG pathway enrichment analysis,the signaling pathways associated with neuroendocrine were significantly enriched in the 2 comparison groups,and in the enriched pathway attribution classification analysis,the neurological system accounted for a greater proportion in PP group compared with the normal group.Venn analysis of differentially expressed proteins in the normal group,PLS group,and CSS group,Slc5a7 protein was discovered.KEGG pathway enrichment analysis of these 10 same differentially expressed proteins,and Cholinergic synapse pathway was enriched.5.m RNA expression of CLOCK,BMAL1,PER1 and PER2 in hippocampus: compared with normal group,CLOCK,BMAL1,PER1 and PER2 expression decreased in LS group and PLS group(P<0.05,P<0.01).Compared with PLS group,the expressions of CLOCK and BMAL1 increased in CSS group(P<0.05).6.Protein levels of hippocampal CLOCK,BMAL1,PER1 and PER2: compared with the normal group,CLOCK,PER1,PER2 and BMAL1 expressions decreased in LS group and PLS group(P<0.01).Compared with PLS group,the expression of CLOCK,BMAL1,PER1 and PER2 increased in PP group(P<0.01),and the expression of CLOCK and PER1 increased in CSS group(P<0.05).Conclusions1.Abnormal neuroendocrine function and abnormal expression of biorhythm genes in rats with PLS pattern lead to disorders of biorhythm.2.CSS can regulate the expression of neuroendocrine and biorhythm genes to improve the disorder of biorhythm,thus improving the performance of PLS pattern in rats.