[Research on mechanism of Panlongqi Tablets regulating β2-adrenergic receptors to improve early fracture injury].

This study aims to clarify the alleviating effects of Panlongqi Tablets(PLQP) on early fracture injuries and to reveal the mechanism of action through a multi-dimensional research method integrating network pharmacology,molecular docking,surface plasmon resonance(SPR),and animal experiments. Firstly,SD rats were randomized into sham operation,model,low-,medium-,and high-dose(0. 16,0. 32,and 0. 64 g·kg~(-1),respectively) PLQP,and positive control(Jiegu Qili Capsules,0. 10 g·kg~(-1)) groups,with 12 rats in each group. Except the sham operation group,other groups were modeled for open transverse fracture of the tibia. After modeling,administration groups received corresponding drugs by gavage for 7 days. The efficacy was comprehensively evaluated in terms of local tenderness,mechanical pain,swelling degree,skin temperature,coagulation function,gait,and histopathological changes of fractures. Then,the fracture transcriptome dataset(GSE262132) from the GEO database and the corresponding targets of the blood-absorbed components of PLQP were collected. By comparison among different time points of fractures,the strongest timeeffect intervention targets of PLQP were predicted,and they were then verified by molecular docking,SPR,and animal experiments. The results showed that PLQP significantly reduced the local swelling and skin temperature in the early stage of fractures,increased the mechanical pain threshold and local tenderness threshold of the affected limb,improved the gait and coagulation function of the affected limb,reduced inflammatory cell infiltration in the callus,lowered the content of fibrous connective tissue,increased the deposition of cartilage and collagen fibers,and promoted angiogenesis in model rats. Network pharmacological analysis indicated that β2-adrenergic receptor(ADRB2),interleukin(IL)-1β,endothelial nitric oxide synthase(NOS3),tumor necrosis factor(TNF),and endothelin-1(EDN1) were candidate strong targets for PLQP intervention in the early stage(7 days) of fractures. Among them,ADRB2 and EDN1 were specific. The results of molecular docking and SPR showed that the blood-absorbed components of PLQP,such as cryptotanshinone,11-keto-β-boswellic acid,tetrahydroberberine,β-ecdysterone,and tectorigenin,all had good binding activities with ADRB2. The results of immunohistochemistry,immunofluorescence,Western blot,and ELISA showed that PLQP upregulated the coexpression of macrophage CD68~+ADRB2~+,osteoblast Osterix~+ADRB2~+,and vascular endothelial cell CD31~+ADRB2~+ in fracture callus,down-regulated the protein level of the M1 macrophage marker i NOS and inhibited the secretion of IL-1β,IL-6,and TNF-α,increased the level of the M2 macrophage marker CD206,and up-regulated the secretion of IL-10. In addition,PLQP increased the expression levels of VEGF and e NOS and the ratio of encoding gene EDN1(ET-1) to nitric oxide(NO). In conclusion,PLQP can significantly alleviate the redness,swelling,heat,and pain symptoms and functional disorders,correct the hypercoagulable state,improve microcirculation,inhibit inflammation,and promote angiogenesis,thereby accelerating fracture healing in rats. Its mechanism of action may be related to the up-regulation of ADRB2 expression for promoting M2-type polarization of macrophages,enhancing VEGF production,and regulating the ET-1/NO balance. The results are helpful in clarifying the therapeutic advantages,characteristics,and scientific connotations of Chinese patent medicines represented by PLQP and guiding the rational use of drugs in clinical practice.