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摘要：
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摘要：目的 探讨黄柏碱（phellodendrine，PHE）通过炎症小体对肝癌细胞增殖和凋
亡的影响。方法 将体外培养的人肝癌细胞系HepG2进行以下处理：空白对照组不添
加任何药物，L-PHE组使用10 μmol/L PHE处理，M-PHE组使用20 μmol/L PHE处理，
H-PHE组使用40 μmol/L PHE处理；阳性对照组使用10 μmol/L顺铂处理。验证PHE调控
NOD样受体蛋白3（NOD-like receptor family pyrin domain containing 3，NLRP3）及下
游信号通路时将HepG2细胞进行以下处理：空白对照组不进行任何干预；si-NC组使用
非靶向序列的干扰小RNA（small interfering RNA，siRNA）进行转染，作为阴性对照；
si-NLRP3组使用针对NLRP3的siRNA进行转染，敲低NLRP3表达；si-NLRP3 + PHE组
先使用针对NLRP3的siRNA进行转染，再用40 μmol/L PHE处理。采用Western blot法检
测NLRP3及含胱氨酸的天冬氨酸蛋白水解酶-1（cysteinyl aspartate specific proteinase-1，
Caspase-1）的相对表达量，采用酶联免疫吸附试验（enzyme-linked immunoadsordent
assay，ELISA）检测肝癌细胞中白细胞介素（interleukin，IL）-1β、IL-6、肿瘤坏死因
子α（tumor necrosis factor α，TNF-α）、IL-8和IL-18等炎症因子的水平。采用CCK-8法检
测肝癌细胞的增殖，采用TUNEL实验检测细胞凋亡。结果 相较于空白对照组，L-PHE
组、M-PHE组、H-PHE组及阳性对照组肝癌细胞中NLRP3（0.82 ± 0.12比0.54 ± 0.06比
0.33 ± 0.04比0.28 ± 0.03比1.07 ± 0.16）、Caspase-1（0.74 ± 0.08比0.51 ± 0.04比0.32 ± 0.02
比0.34 ± 0.04比1.05 ± 0.13）、IL-1β [（59.72 ± 3.58）ng/L比（50.13 ± 4.34）ng/L比（34.43 ±
3.15）ng/L比（35.87 ± 4.42）ng/L比（75.35 ± 4.27）ng/L]、IL-6 [（36.42 ± 3.51）ng/L比（25.85 ±
3.23）ng/L比（17.53 ± 2.82）ng/L比（15.93 ± 2.61）ng/L比（53.23 ± 4.16）ng/L]、
TNF-α [（78.14 ± 5.25）ng/L比（70.33 ± 4.81）ng/L比（55.72 ± 4.67）ng/L比（56.43 ±
4.52）ng/L比（87.91 ± 8.23）ng/L]、IL-8 [（75.16 ± 5.31）ng/L比（62.54 ± 5.12）ng/L
比（47.35 ± 4.93）ng/L比（45.68 ± 4.77）ng/L比（91.42 ± 7.84）ng/L]、IL-18 [（65.35 ± 4.51）ng/L
比（51.83 ± 4.22）ng/L比（37.55 ± 3.18）ng/L比（38.35 ± 3.64）ng/L比（75.56 ± 5.79）ng/L]
水平均显著降低，细胞增殖能力降低，凋亡能力增强（P均＜ 0.05）。与L-PHE组相
比，M-PHE组、H-PHE组及阳性对照组肝癌细胞中NLRP3、Caspase-1及炎症因子水平
均显著降低，细胞增殖能力降低，凋亡能力增强（P均＜ 0.05）。与M-PHE组相比，
H-PHE组及阳性对照组肝癌细胞中NLRP3、Caspase-1及炎症因子水平均显著降低，细
胞增殖能力降低，凋亡能力增强（P均＜ 0.05）。H-PHE组与阳性对照组间上述各指
标差异无统计学意义（P ＞ 0.05）。与空白对照组相比，NLRP3组NLRP3（1.28 ± 0.37
比1.02 ± 0.15）、Caspase-1（1.35 ± 0.35比1.06 ± 0.13）、IL-1β [（95.62 ± 5.82）ng/L
比（80.21 ± 4.56）ng/L]、IL-6 [（72.14 ± 6.21）ng/L比（55.34 ± 4.78）ng/L]、TNF-α
[（113.12 ± 9.67）ng/L比（90.12 ± 7.65）ng/L]、IL-8 [（128.21 ± 11.34）ng/L比（92.45 ±
8.21）ng/L]、IL-18 [（107.56 ± 10.51）ng/L比（76.54 ± 6.34）ng/L] 水平均显著升高
（P均＜ 0.05），si-NLRP3组、NLRP3 + PHE组NLRP3（0.38 ± 0.07比0.36 ± 0.04比
1.02 ± 0.15）、Caspase-1（0.55 ± 0.05比0.59 ± 0.06比1.06 ± 0.13）、IL-1β [（45.43 ± 3.67）ng/L比（42.72 ±
4.42）ng/L比（80.21 ± 4.56）ng/L]、IL-6 [（23.11 ± 2.25）ng/L比（21.34 ± 2.61）ng/L比
（55.34 ± 4.78）ng/L]、TNF-α [（49.12 ± 3.24）ng/L比（50.43 ± 4.52）ng/L比（90.12 ±
7.65）ng/L]、IL-8 [（48.21 ± 4.34）ng/L比（50.38 ± 4.77）ng/L比（92.45 ± 8.21）ng/L]、
IL-18 [（37.25 ± 3.62）ng/L比（39.13 ± 3.51）ng/L比（76.54 ± 6.34）ng/L] 水平显著降低
（P均＜ 0.05）；si-NC组无显著变化（P均＞ 0.05）。与NLRP3组比较，si-NLRP3组、
NLRP3 + PHE组NLRP3、Caspase-1及炎症因子水平显著降低（P均＜ 0.05）。与si-NLRP3
组比较，NLRP3 + PHE组炎症因子水平无显著变化（P ＞ 0.05）。结论 PHE可降低
肝癌细胞中NLRP3的表达并抑制肝癌细胞增殖，促进凋亡。这可能是PHE通过抑制
NLRP3表达进而抑制下游蛋白Caspase-1和炎症因子表达实现的。
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Abstract: Objective To investigate the effects of phellodendron (PHE) on the proliferation
and apoptosis of hepatocellular carcinoma cells via regulating inflammasome. Methods
Human hepatocellular carcinoma cell line HepG2 was cultured in vitro and subjected to
various drug treatments as follows: control group (no treatment), L-PHE group (treated
with 10 μmol/L PHE), M-PHE group (treated with 20 μmol/L PHE), H-PHE group (treated
with 40 μmol/L PHE) and positive control group (treated with 10 μmol/L cisplatin). To
verify the regulatory role of PHE on NOD-like receptor family pyrin domain containing
3 (NLRP3) and its downstream pathways, additional experimental groups were established:
control group (no intervention), si-NC group [transfected with non-targeting small interfering
RNA (siRNA) as a negative control], si-NLRP3 group (transfected with siRNA targeting
NLRP3 to knock down its expression) and si-NLRP3 + PHE group (transfected with siRNA
targeting NLRP3 followed by treatment with 40 μmol/L PHE). Western blot analysis was
used to detect the relative expression of NLRP3 and cysteinyl aspartate specific proteinase-1
(Caspase-1). Enzyme-linked immunoadsordent assay (ELISA) was used to detect the levels
of inflammatory factors including interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α),
IL-8 and IL-18. CCK-8 assay was performed to evaluate proliferation rates and TUNEL assay
was conducted to assess apoptosis. Results Compared with those of control group, the levels
of NLRP3 (0.82 ± 0.12 vs. 0.54 ± 0.06 vs. 0.33 ± 0.04 vs. 0.28 ± 0.03 vs. 1.07 ± 0.16), Caspase-1
(0.74 ± 0.08 vs. 0.51 ± 0.04 vs. 0.32 ± 0.02 vs. 0.34 ± 0.04 vs. 1.05 ± 0.13), IL-1β [(59.72 ± 3.58) ng/L
vs. (50.13 ± 4.34) ng/L vs. (34.43 ± 3.15) ng/L vs. (35.87 ± 4.42) ng/L vs. (75.35 ± 4.27) ng/L],
IL-6 [(36.42 ± 3.51) ng/L vs. (25.85 ± 3.23) ng/L vs. (17.53 ± 2.82) ng/L vs. (15.93 ± 2.61) ng/L
vs. (53.23 ± 4.16) ng/L], TNF-α [(78.14 ± 5.25) ng/L vs. (70.33 ± 4.81) ng/L vs. (55.72 ± 4.67) ng/L vs.
(56.43 ± 4.52) ng/L vs. (87.91 ± 8.23) ng/L], IL-8 [(75.16 ± 5.31) ng/L vs. (62.54 ± 5.12) ng/L
vs. (47.35 ± 4.93) ng/L vs. (45.68 ± 4.77) ng/L vs. (91.42 ± 7.84) ng/L] and IL-18 [(65.35 ±
4.51) ng/L vs. (51.83 ± 4.22) ng/L vs. (37.55 ± 3.18) ng/L vs. (38.35 ± 3.64) ng/L vs. (75.56 ±
5.79) ng/L] reduced significantly in L-PHE group, M-PHE group, H-PHE group and positive
control group (all P ＜ 0.05). Additionally, cell proliferation was inhibited and apoptosis
was enhanced in these groups (all P ＜ 0.05). Compared with those in L-PHE group, the
relative expression of NLRP3 and Caspase-1 and the levels of inflammatory cytokines
reduced significantly in M-PHE group, H-PHE group and positive control group (all P ＜
0.05), while the cell proliferation decreased and cell apoptosis increased (all P ＜ 0.05).
Similarly, compared with the M-PHE group, the H-PHE and positive control group exhibited
significantly lower levels of NLRP3, Caspase-1 and inflammatory cytokine (all P ＜ 0.05), along
with reduced cell proliferation and enhanced cell apoptosis (all P ＜ 0.05). No significant
differences were observed between H-PHE group and positive control group in terms of
NLRP3, Caspase-1 and inflammatory cytokine levels (all P ＞ 0.05). Compared with those of
control group, NLRP3 group showed significantly increased levels of NLRP3 (1.28 ± 0.37 vs.
1.02 ± 0.15), Caspase-1 (1.35 ± 0.35 vs. 1.06 ± 0.13), IL-1β [(95.62 ± 5.82) ng/L vs. (80.21 ±
4.56) ng/L], IL-6 [(72.14 ± 6.21) ng/L vs. (55.34 ± 4.78) ng/L], TNF-α [(113.12 ± 9.67) ng/L vs.
(90.12 ± 7.65) ng/L], IL-8 [(128.21 ± 11.34) ng/L vs. (92.45 ± 8.21) ng/L] and IL-18 [(107.56 ±
10.51) ng/L vs. (76.54 ± 6.34) ng/L], si-NLRP3 group and si-NLRP3 + PHE group showed
significantly reduced levels of NLRP3 (0.38 ± 0.07 vs. 0.36 ± 0.04 vs. 1.02 ± 0.15), Caspase-1
(0.55 ± 0.05 vs. 0.59 ± 0.06 vs. 1.06 ± 0.13), IL-1β [(45.43 ± 3.67) ng/L vs. (42.72 ± 4.42) ng/L vs.
(80.21 ± 4.56) ng/L], IL-6 [(23.11 ± 2.25) ng/L vs. (21.34 ± 2.61) ng/L vs. (55.34 ± 4.78) ng/L], TNF-α
[(49.12 ± 3.24) ng/L vs. (50.43 ± 4.52) ng/L vs. (90.12 ± 7.65) ng/L], IL-8 [(48.21 ± 4.34) ng/L vs.
(50.38 ± 4.77) ng/L vs. (92.45 ± 8.21) ng/L] and IL-18 [(37.25 ± 3.62) ng/L vs. (39.13 ± 3.51) ng/L
vs. (76.54 ± 6.34) ng/L] (all P ＜ 0.05), while no significant changes were observed in
si-NC group (P ＞ 0.05). Compared with those of NLRP3 group, the levels of NLRP3,
Caspase-1 and inflammatory factors in si-NLRP3 group and NLRP3 + PHE group were
significantly reduced (all P ＜ 0.05). Compared with those of si-NLRP3 group, no significant
differences were found in NLRP3, Caspase-1 and inflammatory cytokine levels in si-NLRP3 +
PHE group (P ＞ 0.05). Conclusions PHE could reduce the expression of NLRP3 in
HCC cells, inhibit cell proliferation, and promote apoptosis. This effect may be mediated
through the suppression of NLRP3 expression, which subsequently inhibited the activation
of downstream proteins such as Caspase-1 and the production of inflammatory cytokines.
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