Abstract: BACKGROUND: Abnormal Toll-like receptor 4 (TLR4) signaling pathway plays an important role in the pathogenesis of steroid-induced necrosis of femoral head, and regulating TLR4 expression is expected to become a breakthrough point for effective treatment of hormonal femoral head necrosis.
OBJECTIVE: To study the effect of Tongluo Shenggu Capsule on the TLR4 signal transduction pathway in the process of osteoclast differentiation, in order to understand the molecular biological mechanism by which Tongluo Shenggu Capsule inhibits osteoclast differentiation.
METHODS: Twenty-eight 12-week-old C57BL/6 mice were randomly divided into Tongluo Shenggu Capsule high-dose, medium-dose, low-dose gavage group and normal saline gavage group. Medium dose was set to be 0.91 g/(kg·d), the high dose was 2 times up on the medium dose, and the low dose was 0.5 time up on the medium dose. The drug-containing serum was prepared by continuous gavage for 14 days. At 8 hours after the last administration, blood samples were taken from the abdominal aorta to prepare the serum containing the drug and the control serum. RAW264.7 cell line was induced by the combination of receptor activator of nuclear factor kappa B ligand and macrophage colony stimulating factor inducer. The cells were divided into five groups: normal group, normal saline group, high-, medium-, and low-dose drug-containing serum groups. Cell counting kit-8 method was used to observe the effect of drug-containing serum on cell proliferation. Then, the precursor of osteoclast was pretreated with 20% drug-containing serum on the 4th day of induction. Cell growth, morphology, and fusion were observed at 24, 48, 72, and 96 hours after induction. The number of osteoclasts was observed by TRAP staining on the 8th day. The expression of TLR4 and NF-κBp65 in cell supernatant was detected by western blot. The level of tumor necrosis factor α in cell supernatant was detected using ELISA. The study protocol was approved by the Animal Experimental Ethics Committee of Guangxi University of Chinese Medicine.
RESULTS AND CONCLUSION: The level of tumor necrosis factor α in low-, medium- and high-dose drug-containing serum groups was lower than that in the normal group (P < 0.01), whereas there was no significant difference between the low-dose and medium-dose drug-containing serum groups. The protein expression of TLR4 in low-, medium- and high-dose drug-containing serum groups was significantly lower than that in the normal group (P < 0.05), whereas the protein expression of NF-κBp65 in the medium- and high-dose drug-containing serum groups was significantly lower than that in the normal group (P < 0.05). Moreover, inhibiting the protein expression of TLR4 and NF-κBp65 was most obvious in the medium-dose drug-containing serum group. Overall, one of the mechanisms underlying Tongluo Shenggu Capsule in the treatment of steroid-induced necrosis of femoral head may be to reduce the release of tumor necrosis factor α and improve the inflammatory environment by inhibiting TLR4/NF-κB signal pathway; on the other hand, it can inhibit the activity of osteoclasts, weaken bone absorption and improve the balance of bone metabolism after steroid-induced necrosis of femoral head.

Key words: compound traditional Chinese medicine, factor, bone resorption, osteoclast, Toll-like receptor 4, nuclear transcription factor-B, RAW264.7 cell