JPF promotes repair of early nontraumatic osteonecrosis of the femoral head by inhibiting NAMPT/STK11/HMGCR/ACAT1 signal axis-mediated lipid production

Jianpi Huogu Formula (JPF) has achieved good curative effects in nontraumatic osteonecrosis of the femoral head (NONFH) in clinical practice. However, its underlying pharmacological mechanisms remain unclear. Herein, chemical constituents of JPF were systematically identified by UPLC-Q-TOF-MS, and the putative targets were predicted using TCMIP v2.0 platform. Then, the transcriptomic profiling based on clinical cohorts were carried out to identify the early NONFH-related genes and JPF effective targets. Following network-based calculation and functional enrichment analysis, JPF candidate targets against early NONFH were screened and further validated by a series of in vivo experiments. As a result, a total of 299 chemical constituents in JPF were identified under positive and negative ion mode of mass spectrometry. Pharmacologically, JPF was demonstrated to effectively reduce the content of blood lipids and the number of adipocytes in bone marrow, decrease the serum levels of various inflammatory factors, relieve the joint pain, maintain the normal morphology of bone trabeculae and reduce bone loss of early NONFH rats. Mechanically, JPF markedly suppressed the activities of NAMPT, NMNAT1 and NMNAT3 in the affected femoral head tissues, leading to the reduce of NAD+ synthesis and its mediated ATP synthesis, subsequently promoted the combination of STK11 with energy receptor AMPK, which further reduced the accumulation of cholesterol esters in the liver and the amount of cholesterol esters transported to the blood. In conclusion, JPF may promote the repair of early NONFH with reducing lipid production, improving blood hypercoagulable state and restoring normal bone metabolism by regulating NAMPT/STK11/HMGCR/ACAT1 signal axis. The whole blood samples collected from human in different groups were used to detect the gene expression profiling which may represent the overall status of the body and the pathological characteristics of the affected tissues during early NONFH development and progression by RNA-seq. Differential genes among different groups were identified by the edge R package with Pvalue < 0.05 and fold change >1. Among them, the JPF effective targets (DEGs of JPF pre-treatment group vs. JPF post-treatment group) were identified.The JPF consists of Citrus reticulata Blanco, Paeonia lac tiflor a Pall., Codonopsis pilosula (Franch.) Nannf., Rehmannia glutinosa Libosch., Cinnamomum cassia Presl, Angelica sinensis (Oliv.) Diels, Cyathula officinalis Kuan, Ligusticum chuanxiong Hort., Atractylodes macrocephala Koidz., Poria cocos (Schw.) Wolf, and Deerhorn glue.