ObjectiveTo explore the mechanism of Zhuangyao Tongluo Formula(壮腰通络方,ZTF) in delaying
intervertebral disc degeneration. MethodsM1
macrophages were induced from
THP-1 cells using LPS, IFN-γ and PMA. The induced M1
macrophages were then co-cultured with
nucleus pulposus cells in a transwell system.
Fetal bovine serum was used as the control
serum, and the effects of different concentrations (5%, 10%, 15%, 20%) of
serum from
rats treated with ZTF on the activity of M1
macrophages and
nucleus pulposus cells were analyzed using MTT assay. Experiment 1 was established, including the
nucleus pulposus cell control group, M1
macrophage control group,
nucleus pulposus cell + ZTF group,
nucleus pulposus cell + TNF
control group,
nucleus pulposus cell + TNF + ZTF group,
co-culture group, and
co-culture + ZTF group. The levels of
IL-1β, and
IL-18 in the
culture supernatant were detected using
ELISA. The
mRNA expression of
IL-1β and
IL-18 in
nucleus pulposus cells was detected using qPCR. Additionally, the expression of GSDMD
protein in
nucleus pulposus cells was detected using
cell immunofluorescence. In experiment 2,
co-culture groups were constructed using TNF-α overexpression (OE) or empty vector (EV)
plasmids, including
co-culture group, TNF-EV +
co-culture group, TNF-EV
co-culture group + ZTF,
co-culture + ZTF group, TNF-OE
co-culture group + ZTF, and TNF-OE +
co-culture group. The
mRNA and
protein expression of TNF-α in M1
cells in each group were detected using qPCR and WB. ResultsThe ZTF with 10%
serum was selected for subsequent experiments. The results of experiment 1 showed that compared to the
control group of
nucleus pulposus cells, there was no statistically significant difference in the levels of
IL-1β,
IL-18,
mRNA, and GSDMD expression in the
nucleus pulposus cells + ZTF group (P>0.05). However, the TNF-α +
co-culture group showed a significant increase in
IL-1β,
IL-18 levels,
mRNA, and GSDMD expression (P<0.01). When compared to the
co-culture group, the ZTF+
co-culture group showed a significant decrease in
IL-1β,
IL-18 levels,
mRNA, and GSDMD expression (P<0.01). The results of experiment 2 showed that there was no statistically significant difference in TNF-α
mRNA and
protein expression between the empty vector
plasmids +
co-culture group and the
co-culture group (P>0.05). Compared to the empty vector +
co-culture group, the expression of TNF-α
mRNA and
protein was significantly reduced in the empty vector
co-culture + ZTF group (P<0.01). Compared to the
co-culture group and the empty vector +
co-culture group, the expression of TNF-α
mRNA and
protein was significantly reduced in the
co-culture + ZTF group (P<0.01). Compared to the
co-culture + ZTF group, the expression of TNF-α
mRNA and
protein significantly increased in the overexpression vector
co-culture + ZTF group (P<0.01). Compared to the overexpression vector
co-culture + ZTF group, the expression of TNF-α
mRNA and
protein significantly increased in the overexpression vector
co-culture group (P<0.01). ConclusionZTF
serum can inhibit the TNF-α-induced
apoptosis of
nucleus pulposus cells and delay lumbar
disc degeneration by reducing the expression of TNF-α in M1
macrophages.