Abstract: BACKGROUND: Lumbar disc degeneration is the main pathological change of lumbar disc herniation, which is closely related to the programmed death of intervertebral disc annulus fibrosus cells. Previous clinical studies have shown that Tongdu Huoxue Decoction has a significant effect in the treatment of lumbar disc herniation, and it is worth further studying whether it can play a role by inhibiting the pyrolysis of annulus fibrosus cells. 
OBJECTIVE: To investigate the inhibitory effect of Tongdu Huoxue Decoction-medicated serum on the pyroptosis of annulus fibrosus cells in the intervertebral disc induced by lipopolysaccharide/adenosine triphosphate (ATP), so as to explore the possible mechanism of Tongdu Huoxue Decoction in the treatment of lumbar disc herniation. 
METHODS: Thirty-six 10-week-old Spraue-Dawley rats were gavaged with Tongdu Huoxue Decoction, and blood samples were taken from the abdominal aorta after 7-day continuous gavage to prepare drug-containing serum. Four-week-old Sprague-Dawley rats were taken to extract their lumbar intervertebral discs, and then the annulus fibrosus cells were extracted from the intervertebral disc tissues by mechanical-enzymatic digestion. The cells cultured to the second generation were identified by toluidine blue staining and type II collagen immunohistochemical staining. After that, the second-generation cells were randomly divided into blank group, model group, and low-, middle-, and high-dose serum-containing groups. Except for the blank group, the remaining four groups were given lipopolysaccharide/ATP to make the cell pyroptosis model. After modeling, 5%, 10%, and 20% of Tongdu Huoxue Decoction medicated serum were administrated in the low-, middle-, and high-dose serum-containing groups, respectively, and no intervention measures were taken in the blank group. We then used the MTT method to find the best concentration and time for the intervention of pyroptosis of annulus fibrosus cells with the drug-containing serum. Flow cytometry and Annexin V FIFT/PI double staining were used to measure the rate of cell pyroptosis. ELISA method was used to detect the expression of interleukin-1β and interleukin-18 in cell supernatant. qRT-PCR was used to detect the mRNA expression of Caspase-1, Gasdermin D, and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3). 
RESULTS AND CONCLUSION: MTT showed that the best intervention concentration of Tongdu Huoxue Decoction medicated serum for the pyroptosis of annulus fibrosus cells was 10%, and the best intervention time was 24 hours. Flow cytometry results showed that compared with the blank group, the pyroptosis rate in the other four groups was significantly increased (P < 0.05). Compared with the model group, the pyroptosis rate in the three drug-containing serum groups was significantly decreased (P < 0.05). Compared with the low- and high-dose groups, the pyroptosis rate of the middle-dose group was significantly decreased (P < 0.05). Results from ELISA, qRT-PCR, and western blot assay indicated that compared with the blank group, the expression levels of interleukin-1β, interleukin-18, Caspase-1, Gasdermin D, NLRP3 mRNAs and proteins were significantly increased (P < 0.05), while compared with the model group, these expression levels were significantly decreased in the three drug-containing serum groups (P < 0.05). Moreover, the expression levels of interleukin-1β, interleukin-18, Caspase-1, Gasdermin D, NLRP3 in the middle-dose group were significantly lower than those in the low- and high-dose groups (P < 0.05). Therefore, Tongdu Huoxue Decoction-medicated serum can effectively relieve lipopolysaccharide/ATP induced pyroptosis of intervertebral disc annulus fibrosus cells and reduce inflammation. The middle-dose group has the best effect in delaying cell pyroptosis and reducing inflammation.

Key words: Tongdu Huoxue Decoction, medicated serum, lumbar disc herniation, annulus fibrosus cells, pyroptosis