Effect And Mechanism Of Paraventricular Nucleus Pathway In Substantia Nigra On Brown Adipose Tissue Thermogenesis In Rats

BackgroundThe paraventricular nucleus(PVN)of the hypothalamus can regulate fat metabolism through the sympathetic nervous system(SNS).The brain-derived neurotrophic factor(BDNF)positive neurons in PVN can regulate the adaptive thermogenesis of interscapular brown adipose tissue(IBAT),but its upstream regulation mechanism is still unclear.Fat loss is an important cause of weight loss in Parkinson's disease(PD)patients,but the mechanism is unknown.A large number of dopaminergic(DA)neurons in the substantia nigra(SN)of PD patients were lost,accompanied by decreased PVN function and enhanced SNS activity.Our previous research found that a large number of dopamine receptors(DRs)are expressed on PVN,and the SN can directly project to PVN(SN-PVN pathway).Therefore,we hypothesized that the upstream pathway regulating BDNF expression in PVN might be DRs-mediated SN-PVN pathway.ObjectivesThe purpose of this study is to determine whether dopamine in SN affects brown fat metabolism,to determine the co-localization relationship between BDNF and DRS in PVN,and to determine whether the SN-PVN pathway can be used as an upstream pathway for BDNF to regulate brown fat thermogenesis.And to explore the role of DRs in the regulation of IBAT thermogenesis of the SN-PVN pathway,provide theoretical and experimental basis for DA to regulate fat metabolism,and provide references for revealing the relationship between DA and obesity and other metabolic diseases.Methods1.The model was established by stereotactic technique: the rats were divided into normal group,6-OHDA group and control group;the normal group was not treated;in the6-OHDA group,6-OHDA was injected into bilateral SN for damage of DAergic neurons,and in the control group,0.2% ascorbic acid was injected into SN for control.2.Rats in the control group and 6-OHDA group were weighed every week after surgery.After successful modeling,food intake of rats was measured and IBAT was weighed.3.Nissl staining was used to localize the SN,PVN and IML nuclei.4.The immunofluorescence staining was used to detect TH expression in SN and Ch AT and BDNF receptor Trk B expression in the IML of the spinal cord.5.The co-expression location of BDNF and TH,BDNF and D1,BDNF and D2 in PVN and expression changes of BDNF,D1,D2 and TH were detected by immunofluorescence double-standard staining.6.Western blot(WB)was used to detect the protein expression changes of TH in SN and TH,D1,D2 and BDNF in hypothalamus,as well as the protein expression changes of anabolism index fatty acid synthase(FAS),catabolism index phosphorylated hormone-sensitive lipase(p-HSL)and hormone-sensitive lipase(HSL),thermogenesis index uncoupling protein-1(UCP-1)and related indicators of sympathetic nerve activity TH and adrenergic ?3 receptor(?3-AR)in IBAT.7.The IBAT was stained by hematoxylin-Eosin(HE)staining and the adipocyte diameter was measured.Results1.The results of immunofluorescence single label staining and WB showed that the number of TH positive neurons and nerve fibers in SN of rats in 6-OHDA group was significantly reduced compared with the control group,and the protein expression content of TH was significantly reduced.2.The weight and food consumption of the rats were tested,and the results showed that compared with the control group,the postoperative weight and food consumption of the rats in the 6-OHDA model group were reduced,but there was no statistical difference.The IBAT of rats were weighed,and the results showed that compared with the control group,the 6-OHDA group showed a slightly decrease in the IBAT,but there was no statistical difference.3.The results of WB showed that IBAT anabolic indicators FAS decreased,catabolic indicators HSL no changed and p-HSL increased,thermogenic indicators UCP-1 increased,sympathetic nerve activity related indicators TH and ?3-AR increased.IBAT was stained with HE and the diameter of adipocytes was measured.The results showed that the diameter of IBAT adipocytes in 6-OHDA group was smaller than that in control group.4.The results of immunofluorescence single label staining showed that Trk B receptor expression existed in IML.Compared with the control group,Ch AT positive neurons in the6-OHDA group increased in area.5.The results of immunofluorescence double labeling staining showed that D1,D2 and BDNF positive neurons almost coexisted in PVN of normal rats.6.The results of immunofluorescence single and double staining showed that TH positive nerve fibers in PVN of normal rats wrapped around BDNF positive neurons in dots or short strips.7.The results of immunofluorescence double labeling staining and WB showed that compared with the control group,the expression of D1 positive neurons and protein in PVN of 6-OHDA group had no significant change,but the expression of D2 positive neurons and protein decreased significantly.8.The results of immunofluorescence double labeling staining and WB showed that compared with the control group,the expression of BDNF positive neurons and protein in PVN of 6-OHDA group was significantly increased,while the expression of TH positive nerve fibers and protein was significantly decreased.ConclusionsThe BDNF positive neurons in PVN of normal rats almost completely coexisted with D1 and D2 and were surrounded by TH positive nerve fibers.After 6-OHDA-induced bilateral SN damage in rats,although the body weight,IBAT weight and food intake did not significantly change,the decreased expression of D2 and TH in PVN may be the reason for the increased expression of BDNF,and then the increased thermogenesis and catabolism of IBAT,as well as the decreased anabolism and cell diameter.The SN-PVN pathway may be the upstream pathway of BDNF regulating the thermogenesis of IBAT.