Abstract: BACKGROUND: Inflammation and apoptosis play key roles in the pathological process of cervical spondylotic myelopathy. Previous studies have shown that Pueraria lobata decoction has favorable therapeutic effects on cervical spondylotic myelopathy.
OBJECTIVE: To investigate the effects and mechanism of Pueraria lobata decoction in neuroinflammatory response and apoptosis in rats with cervical spondylotic myelopathy. 
METHODS: Sixty Sprague-Dawley rats were randomly divided into six groups: a normal group, a sham-operated group, a model group, and three groups that received low, medium, and high doses of Pueraria lobata decoction. An animal model of cervical spondylotic myelopathy was constructed through compression of the spinal cord with water-absorbing and expanding material. Gastric administration of Pueraria lobata decoction (4.86, 9.72, and 19.44 g/kg) was given in the three Pueraria lobata decoction groups 2 weeks after surgery, and the resting groups were given saline by gavage, once daily for 4 weeks. Motor function evaluation (Basso-Beattie-Bresnahan score) was performed in rats on days 1, 7, 14, 21 and 28 after drug administration. At 4 weeks after drug administration, hematoxylin-eosin staining was used to observe the pathomorphologic changes in spinal cord tissue; immunofluorescence double staining was used for the detection of microglial cell polarization in spinal cord tissue; quantitative fluorescence PCR was used to detect the changes in the expression of interleukin-6 and interleukin-1β mRNA; western blot assay was used to detect the protein expression of p-NF-κB p65, NF-κB p65, NLRP3, ASC, Cleaved Caspase-1, Bax, Bcl-2, Cleaved Caspase-3, NOX4, p-Drp1, Drp1, and Mfn2 in spinal cord tissues; TUNEL assay was used to detect apoptosis in spinal cord tissues; and DHE staining was used to detect reactive oxygen species levels in rat spinal cord tissues.   
RESULTS AND CONCLUSION: (1) Compared with the normal and sham-operated groups, reduced Basso-Beattie-Bresnahan scores were observed in the model group (P < 0.05), spinal cord neurons were crumpled and malformed with vacuolike changes. The Basso-Beattie-Bresnahan scores in the low-, medium- and high-dose Pueraria lobata decoction groups were significantly higher than those in the model group (P < 0.05), and spinal cord neuronal damage reduced significantly. (2) Compared with the normal and sham-operated groups, there were elevated levels of Iba-1 and inducible nitric oxide synthase proteins and increased interleukin-6 and interluekin-1β mRNA expression in the spinal cord tissue of rats in the model group (P < 0.05). The expression levels of Iba-1, inducible nitric oxide synthase, p-NF-κB, NLRP3, ASC and cleaved caspase-1 proteins as well as interleukin-6 and interleukin-1β mRNAs in the spinal cord tissues of rats in the low-, medium- and high-dose Pueraria lobata decoction groups were reduced compared with those in the model group (P < 0.05). (3) Compared with the normal and sham-operated groups, the rate of TUNEL-positive cells and the levels of Bax and cleaved caspase-3 proteins were increased in the spinal cord tissues of rats in the model group (P < 0.05), while the expression of Bcl-2 protein was reduced (P < 0.05). Compared with the model group, the above indexes were significantly improved in the low-, medium- and high-dose Pueraria lobata decoction groups. (4) Compared with the normal and sham-operated groups, the model group exhibited increased levels of reactive oxygen species, along with elevated expression of NOX4 and p-Drp1 proteins (P < 0.05) and reduced expression of Mfn2 protein (P < 0.05) in the rat spinal crod tissue. Compared with the model group, the low-, medium-, and high-dose Pueraria lobata decoction groups exhibited reduced levels of reactive oxygen species, as well as decreased expression of NOX4 and p-Drp1 proteins (P < 0.05) and increased expression of Mfn2 protein (P < 0.05) in the rat spinal cord tissue. To conclude, Pueraria lobata decoction inhibits neuroinflammatory responses and neuronal apoptosis in the rat model of cervical spondylotic myelopathy, and the mechanism of action may be related to the regulation of the NOX4/reactive oxygen species/DRP1 signaling pathway.

Key words: Pueraria lobata decoction, cervical spondylotic myelopathy, spinal cord tissue, microglia, apoptosis, inflammation, NOX4, reactive oxygen species, Drp1, engineered tissue construction