Bushen Tongshi Pills improves osteogenic disorders in alcoholic femoral head necrosis rats

Abstract

BACKGROUND: Bushen Tongshi Pills has been proven to delay the progression of collapse in alcoholic femoral head necrosis, but the mechanism is still unclear. OBJECTIVE: To explore the mechanism by which Bushen Tongshi Pills improve osteogenic disorders of alcoholic femoral head necrosis. METHODS: Fifty male Sprague-Dawley rats were randomly divided into a control group, a model group, and low, medium, and high dose groups of Bushen Tongshi Pills, with 10 rats in each group. Except for the control group, other groups were fed with ethanol-containing Lieber-DeCarli liquid diet for 8 weeks to construct alcoholic femoral head necrosis models, while the low, medium, and high dose groups were given 1.05, 2.1, 4.2 g/kg Bushen Tongshi Pills by gavage daily. After 8 weeks, Micro-CT was used to observe the gross morphology of the femoral head, hematoxylin-eosin staining was used to observe the pathological morphology of the femoral head, ELISA was used to detect serum levels of interleukin-1β and interleukin-18, immunohistochemical staining and western blot were used to detect the expression of NOD-like receptor protein 3, Caspase-1, gasdermin D, Runt-related transcription factor 2, osteocalcin, and type I collagen in rat femoral head tissues, and RT-qPCR was used to detect the mRNA expression of these genes. RESULTS AND CONCLUSION: (1) Micro-CT and hematoxylin-eosin staining results showed that the medium and high dose groups of Bushen Tongshi Pills significantly improved bone loss and pathological morphological development in the femoral head of alcoholic femoral head necrosis model rats. (2) ELISA results showed that compared with the control group, serum levels of interleukin-1β and interleukin-18 in the model group were significantly increased (P < 0.05); compared with the model group, serum levels of interleukin-1β and interleukin-18 in all Bushen Tongshi Pills dose groups were significantly decreased (P < 0.05), in a dose-dependent manner, with the most significant decrease in the high dose group (P < 0.05). (3) Immunohistochemistry, western blot, and RT-qPCR results showed that compared with the control group, the protein and mRNA expression of NOD-like receptor protein 3, Caspase-1, and gasdermin D in the femoral head of model rats were significantly upregulated (P < 0.05), while the protein and mRNA expression of osteogenic factors Runt-related transcription factor 2, osteocalcin, and type I collagen were significantly downregulated (P < 0.05); compared with the model group, the protein and mRNA expression of NOD-like receptor protein 3, Caspase-1, and gasdermin D in the femoral head of rats in all Bushen Tongshi Pills dose groups were downregulated in a dose-dependent manner, while the protein and mRNA expression of Runt-related transcription factor 2, osteocalcin, and type I collagen were upregulated in a dose-dependent manner, with the most significant upregulation in the high dose group (P < 0.05). (4) Western blot results showed that compared with the control group, the expression of cleaved-Caspase-1, gasdermin D-N protein, cleaved-Caspase-1/Caspase-1 ratio, and gasdermin D-N/gasdermin D ratio in the femoral head of model rats were significantly upregulated (P < 0.05); compared with the model group, the expression of cleaved-Caspase-1, gasdermin D-N protein, and gasdermin D-N/gasdermin D ratio in all Bushen Tongshi Pills dose groups were significantly downregulated (P < 0.05), and the cleaved-Caspase-1/Caspase-1 ratio in the medium and high dose groups was significantly downregulated (P < 0.05). (5) These results indicate that Bushen Tongshi Pills may inhibit the release of inflammatory factors in alcoholic femoral head necrosis model rats through the NOD-like receptor protein 3/Caspase-1/gasdermin D pyroptosis pathway, promote osteogenic differentiation, and repair necrotic bone tissue.

1. Introduction

Femoral head necrosis is characterized by reduced blood supply to the femoral head, leading to osteocyte necrosis, trabecular bone fracture, and joint surface collapse [1-2]. Alcoholic femoral head necrosis is a common non-traumatic type, primarily caused by excessive alcohol intake. Abnormal alcohol metabolism can produce various toxic substances, such as acetaldehyde, free radicals, and acetaldehyde adducts, resulting in femoral head tissue damage [3-5].

Pyroptosis is a form of programmed cell death mediated by gasdermins, lying between apoptosis and necrosis [6]. The gasdermin family includes gasdermin A, B, C, D, and E, among which gasdermin D activation is associated with inflammasomes, while others are activated by inflammasome-independent pathways [7]. Inflammasome activation is the initiating event of pyroptosis. Upon activation, inflammasomes lead to self-cleavage and activation of cysteinyl aspartate specific proteinase (Caspase), which then cleaves gasdermin D, releasing its N-terminal fragment (gasdermin D-N). This fragment forms membrane pores, causing leakage of cellular contents, cell lysis, and local inflammation [8-9]. Known inflammasomes include the absent in melanoma 2 inflammasome, the interleukin-1β converting enzyme protease-activating factor inflammasome, the NOD-like receptor protein 1 inflammasome, the NOD-like receptor protein 3 (NLRP3) inflammasome, and the NOD-like receptor family CARD domain-containing protein 4 inflammasome. Among these, the NLRP3 inflammasome is the most extensively studied and is implicated in various inflammatory diseases.