Chaihu-Shugan-San Ameliorated Osteoporosis of Mice with Depressive Behavior Caused by Chronic Unpredictable Mild Stress via Repressing Neuroinflammation and HPA Activity

Ming-Chao He,1,2,* Shi-Hui Xia,2,3,* Hao Pan,4,* Ting-Ting Zhou,5 Xin-Luan Wang,6 Ji-Ming Li,6 Xiao-Ming Li,5 Yan Zhang2,3 1Department of Pain Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, People’s Republic of China; 2Spine Disease Research Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, People’s Republic of China; 3Key Laboratory of Theory and Therapy of Muscles and Bones, Ministry of Education, Shanghai, 200032, People’s Republic of China; 4Department of Neurosurgery, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China; 5Experimental Research Center, Cangzhou Hospital of Integrated TCM-WM, Cangzhou, 061001, People’s Republic of China; 6Translational Medicine R&D Center, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yan Zhang, Longhua Hospital, affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, People’s Republic of China, Tel +86 21-64385700, Email [email protected] Xiao-Ming Li, Cangzhou Hospital of Integrated TCM-WM, Cangzhou, 061001, People’s Republic of China, Tel +86 317-2178055, Email [email protected]: Depression and osteoporosis are usually concurrent health problems. This study aimed to explore the development of osteoporosis in depressive mice model and investigate the beneficial effects of the classical herbal formula Chaihu-Shugan-San (CHSG) on the brain and bone.Methods: CHSG powder was prepared by spray-drying following extraction with water. The fingerprint of CHSG was analyzed using liquid chromatography. The depressive-like model was established by chronic unpredictable mild stress (CUMS) in female mice. The depressive behaviors and trabecular bone properties (measured by micro-CT) were detected at 2, 4, 6, and 8 weeks of CUMS. RT-PCR, immunoblotting and immunofluorescence were applied to measure expression of inflammatory cytokines and morphology of microglias in the hippocampus. Biochemical measurements and histological staining on the adrenal gland were carried out to assess the activity of hypothalamic-pituitary-adrenal (HPA) axis. Histological staining, three-point bending strength, and the expression of regulators involved in bone metabolism were determined.Results: The treatment with CHSG for 8 weeks could ameliorate depressive behaviors, and down-regulate mRNA expression and tissue content of inflammatory factors IL-1β and IL-6 in hippocampus of CUMS mice. The inhibition of CHSG on neuroinflammation might be attributed to its repression of activity in microglias and NLRP3-triggered inflammation pathway. The serum of rats dramatically alleviated LPS-induced phosphorylation of nuclear NFκB (P65) and IκBα and up-regulation of IL-1β and IL-6 proteins in microglia BV2 cells. CUMS induced over-activity of HPA axis shown by the elevation in serum level of ACTH and corticosterone and in area percentage of zona fasciculata, intriguingly, CHSG reversed those changes in HPA system, ameliorated the reduction in mechanical strength and bone mineral density, and regulated bone metabolism factors of CUMS mice.Conclusion: The chronic stress-induced depression resulted in bone disorders developing to osteoporosis. Chaihu-Shugan-San exerted beneficial effects on skeletal tissue by ameliorating neuroinflammation and HPA over-activity of mice with depression. Keywords: Chaihu-Shugan-San, depression, hippocampus, HPA, osteoporosis, stress