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Propofol upregulates MFG‑E8 in BV2 cells to inhibit pyroptosis mediated by the NF‑κB/NLRP3 pathway, thereby ameliorating ischemic‑reperfusion neuronal injury

  • Authors:
    • Shewei Guo
    • Yingwei Zhen
    • Guosheng Zhou
    • Zhihua Zhao
  • View Affiliations / Copyright

    Affiliations: Department of Neurosurgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 453001, P.R. China, Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 453001, P.R. China
    Copyright: © Guo et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 115
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    Published online on: March 5, 2026
       https://doi.org/10.3892/ijmm.2026.5786
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Abstract

Abnormal activation and pyroptosis of microglia caused by cerebral ischemia‑reperfusion injury (CIRI) are key mechanisms underlying neuronal damage. The NF‑κB/NLRP3 pathway is a core mediator of microglial pyroptosis and neuroinflammatory cascades in CIRI. Milk fat globule‑EGF factor 8 (MFG‑E8) is a critical anti‑inflammatory and neuroprotective factor. Propofol (PPF) exhibits antioxidant activity and ameliorates neuronal injury, but its effects on CIRI and underlying mechanisms remain unclear. The present study aimed to investigate whether PPF alleviates neuronal injury by modulating NF‑κB/NLRP3 pathway via regulating MFG‑E8 expression. An oxygen‑glucose deprivation/reoxygenation (OGD/R) model was established using mouse microglial BV‑2 and hippocampal neuronal HT22 cells and cell survival was assessed via Cell Counting Kit‑8 assay. Polarity in BV‑2 cells was evaluated using flow cytometry, while cell death was assessed by Calcein AM/PI and TUNEL staining. A transient middle cerebral artery occlusion (tMCAO) mouse model was established and neurological deficit scores were assessed. The impacts of PPF on cortical damage, neuroinflammation, apoptosis and pyroptosis in tMCAO mice were observed by histopathological staining. Inflammatory factor levels were assessed using ELISA kits. Western blotting was performed to assess MFG‑E8, pyroptosis and NF‑κB/NLRP3 pathway‑related proteins. OGD/R decreased viability, increased apoptosis and pyroptosis rates in BV‑2 and HT22 cells and promoted M1 polarization in BV‑2 cells; PPF treatment reversed these effects. MFG‑E8 was downregulated in OGD/R‑treated BV2 cells, while PPF upregulated MFG‑E8 expression. Additionally, PPF decreased cerebral infarction volume in tMCAO mice, improved neurological deficit score, mitigated pathological brain tissue damage and decreased the number of degenerating neurons. PPF also inhibited pro‑inflammatory microglia activation and decreased pro‑inflammatory factor levels. Mechanistically, PPF suppressed NF‑κB pathway activation and downregulated NLRP3 by upregulating MFG‑E8; silencing MFG‑E8 reduced the protective effects of PPF in tMCAO mice and OGD/R cell models. PPF improved neuronal injury in CIRI by upregulating MFG‑E8 to inhibit pyroptosis induced by the NF‑κB/NLRP3 pathway.
View Figures

Figure 1

PPF suppresses excessive activation
of BV-2 cells and ameliorates damage in HT22 cells following OGD/R.
(A) Operational flowchart. (B) Cell Counting Kit-8 assay was
performed to determine the viability of BV2 and HT22 cells after 24
h treatment with PPF. (C) Flow cytometry revealed that PPF
decreased the number of CD86-positive and increased the number of
CD206-positive cells. (D) Quantitative analysis of flow cytometry
confirmed that PPF significantly decreased the proportion of
CD86-positive cells and (E) significantly increased the proportion
of CD206-positive cells. (F) Western blotting of iNOS, Arg 1, and
GAPDH (internal reference). (G) Western blotting revealed that PPF
decreased iNOS and increased Arg 1 levels in BV-2 cells. (H)
Calcein AM/PI staining revealed decreased viability in
OGD/R-treated HT22 cells, which was diminished upon co-culture with
BV-2 cells, whereas PPF enhanced cell survival (magnification, ×20;
scale bar, 100 μm). (I) Quantitative analysis of Calcein
AM/PI staining showed that PPF enhanced the survival ability of
HT22 cells in the OGD/R+BV2 co-culture system and (J) decreased the
death rate of HT22 cells in the OGD/R+BV2 co-culture system. (K)
TUNEL staining to assess TUNEL positivity in HT22 cells (green
fluorescence) (magnification, ×40; scale bar, 50 μm). (L)
TUNEL staining indicated increased apoptosis in OGD/R-treated HT22
cells. Co-culture with BV-2 cells elevated apoptosis rates, whereas
PPF reduced apoptosis. (M) Following OGD/R, LDH release increased
in HT22 cells and was further elevated after co-culture with BV-2
cells, while PPF treatment decreased LDH release. (N) Western
blotting protein bands for SYP-1, PSD-95, BDNF, GAP-43, and GAPDH.
(O) Quantitative analysis of western blotting revealed that PPF
induced elevated SYP-1, PSD-95, BDNF and GAP-43 levels in HT22
cells. *P<0.05, **P<0.01. PPF,
Propofol; OGD/R, Oxygen-glucose deprivation/reoxygenation; iNOS,
inducible nitric oxide synthase; Arg, arginase 1; LDH, lactate
dehydrogenase; SYP, synapsin; PSD, postsynaptic density protein;
BDNF, brain-derived neurotrophic factor; GAP, growth-associated
protein; FCM, flow cytometry.

Figure 2

PPF suppresses NLRP3-mediated
pyroptosis in BV2 cells following OGD/R. (A) SEM observation of BV2
cell morphology (magnification, ×20,000; scale bar, 2 μm).
(B) Yo-Pro-1 and Hoechst 33342 staining for assessing the
pyroptosis levels in BV-2 cells. (C) Quantitative analysis of
Yo-Pro-1 and Hoechst 33342 staining revealed that PPF deceased
pyroptosis levels in BV-2 cells. (D) Immunofluorescence staining of
NLRP3 in BV2 cells (magnification, ×40; scale bar, 50 μm).
(E) Immunofluorescence staining of ASC in BV2 cells (magnification,
×40; scale bar, 50 μm). (F) Quantitative analysis of
immunofluorescence staining revealed that NLRP3 and ASC
fluorescence intensity increased in OGD/R-treated BV2 cells, while
PPF decreased the fluorescence intensity of both proteins. (G)
Caspase-1 activity was detected in BV2 cells following OGD/R using
Caspase-1 Activity Assay kit. (H) Western blotting of NLRP3, ASC,
cleaved-caspase-1, pro-caspase-1, GSDMD-N, GSDMD, IL-18, IL-1β, and
GAPDH (internal reference). (I) Western blot analysis revealed
elevated NLRP3, ASC, IL-1β and IL-18,
cleaved-caspase-1/pro-caspase-1 levels (J), and GSDMD-N/GSDMD
levels (K) in OGD/R-treated BV2 cells, PPF treatment reduced these
proteins levels. (L) ELISA showed that PPF decreased IL-1β and
IL-18 levels. **P<0.01. PPF, Propofol; OGD/R,
Oxygen-glucose deprivation/reoxygenation; ASC, apoptosis-associated
speck-like protein containing a CARD; GSDMD, gasdermin D.

Figure 3

Overexpression of MFG-E8 suppresses
NF-κB and NLRP3 activation in OGD/R-induced BV2 cells. (A) Western
blotting protein bands for MFG-E8, p-NF-κB, NF-κB, and GAPDH
(internal reference). (B and C) Following OGD/R, western blotting
revealed decreased MFG-E8 expression (B) and elevated p-NF-κB/NF-κB
levels (C) in BV2 cells. (D) Western blotting protein bands for
MFG-E8 and GAPDH (internal reference). (E) Following transfection
of OE-MFG-E8 into BV2 cells, MFG-E8 expression was elevated
markedly. (F) Western blotting protein bands for NLRP3, ASC,
p-NF-κB, NF-κB, and GAPDH (internal reference). (G and H) Following
transfection with OE-MFG-E8, p-NF-κB/NF-κB levels (G), and NLRP3
and ASC levels (H) were detected using western blotting. (I)
Western blotting protein bands for NLRP3, ASC, p-NF-κB, NF-κB, and
GAPDH (internal reference). Following treatment with the NF-κB
activator LPS, p-NF-κB/NF-κB (J), and NLRP3 and ASC levels (K) were
detected by western blotting. (L) Western blotting protein bands
for p-NF-κB, NF-κB, and GAPDH (internal reference). (M) Following
transfection with OE-MFG-E8, p-NF-κB/NF-κB expression was
decreased, whereas treatment with LPS increased p-NF-κB/NF-κB
levels. (N) Immunofluorescence staining of NLRP3 in BV2 cells
(magnification, ×40; scale bar, 50 μm). (O)
Immunofluorescence staining of ASC in BV2 cells (magnification,
×40; scale bar, 50 μm). (P) Immunofluorescence revealed
OE-MFG-E8 reduced the fluorescence intensity of NLRP3 and ASC,
while LPS diminished the effect of MFG-E8 overexpression. (Q)
Western blotting protein bands for NLRP3, ASC, cleaved-caspase-1,
pro-caspase-1, GSDMD-N, GSDMD, IL-1β, IL-18, and GAPDH (internal
reference). (R-T) Western blotting revealed that OE-MFG-E8 reduced
cleaved-caspase-1/pro-caspase-1 levels (R), GSDMD-N/GSDMD levels
(S), NLRP3, ASC, IL-1β, and IL-18 levels (T) in OGD/R-induced BV2
cells, LPS attenuated the impact of OE-MFG-E8.
**P<0.01. PPF, Propofol; OGD/R, Oxygen-glucose
deprivation/reoxygenation; ASC, apoptosis-associated speck-like
protein containing a CARD; MFG-E8, milk fat globule-EGF factor 8;
GSDM, gasdermin; OE, overexpression; p-, phosphorylated; LPS,
lipopolysaccharide; NC, negative control; ns, not significant.

Figure 4

PPF suppresses pyroptosis caused by
NF-κB/NLRP3 signaling by upregulating MFG-E8. (A) Western blotting
protein bands for MFG-E8, p-NF-κB, NF-κB, and GAPDH (internal
reference) in BV2 cells. (B) Western blotting revealed that PPF
increased MFG-E8 and decreased p-NF-κB/NF-κB levels (C) in BV2
cells. (D) Western blotting for MFG-E8 and GAPDH in BV2 cells. (E)
Following transfection of si-MFG-E8 into BV2 cells, MFG-E8 protein
levels were significantly decreased. (F) Dual luciferase reporter
gene assay confirmed that NF-κB is the target of MFG-E8. (G)
Western blotting protein bands for p-NF-κB, NF-κB, and GAPDH
(internal reference) in BV2 cells. (H) si-MFG-E8 attenuated the
effects of PPF, resulting in elevated p-NF-κB/NF-κB levels. (I)
Yo-Pro-1 and Hoechst 33342 staining for assessing the pyroptosis
levels in BV-2 cells (magnification, ×40; scale bar, 50 μm).
(J) Yo-Pro-1 and Hoechst 33342 staining showed that PPF decreased
Yo-Pro-1 positivity, while silencing MFG-E8 increased Yo-Pro-1
positivity. ELISA showed that PPF decreased TNF-α (K) and IL-1β (L)
levels, silencing MFG-E8 reversed this effect. (M and N) ELISA
showed that PPF raised IL-10 levels (M) and decreased IL-6 levels
(N), silencing MFG-E8 reversed this effect. (O) Western blotting
protein bands for NLRP3, ASC, cleaved-caspase-1, pro-caspase-1,
GSDMD-N, GSDMD, IL-1β, IL-18, and GAPDH (internal reference) in BV2
cells. (P-R) Western blot analysis indicated that PPF decreased
NLRP3, ASC, IL-1β, and IL-18 levels (P), GSDMD-N/GSDMD levels (Q),
cleaved-caspase-1/pro-caspase-1 levels (R), silencing MFG-E8
increased these protein levels. **P<0.01. PPF,
propofol; MFG-E8, milk fat globule-EGF factor 8; OGD/R,
Oxygen-glucose deprivation/reoxygenation; ASC, apoptosis-associated
speck-like protein containing a CARD; GSDM, gasdermin; OE,
overexpression; p-, phosphorylated; LPS, lipopolysaccharide; NC,
negative control; ns, not significant; si, small interfering; WT,
wild-type; MUT, mutant.

Figure 5

PPF improves neurological deficit and
decreases brain tissue pathology in tMCAO mice. (A) Operational
flowchart. (B) Longa scoring indicated that PPF decreased
neurological deficit scores in tMCAO mice (n=9). (C) Corner test
results demonstrated that PPF decreased sensorimotor deficits in
tMCAO mice. (D) PPF decreased brain water content in tMCAO mice.
(E) TTC staining to assess cerebral infarct lesions in tMCAO mice.
(F) TTC staining revealed well-defined infarct lesions in the
cerebral cortex of tMCAO mice, with PPF diminishing infarct volume.
(G) HE staining was used to assess the histopathological brain
damage in tMCAO mice (magnification, ×40; scale bar, 50 μm).
(H) Nissl staining was used to assess the proportion of
Nissl-positive cells in the brain tissue of tMCAO mice
(magnification, ×40; scale bar, 50 μm). (I) Nissl staining
revealed a decrease in Nissl-positive cell count in tMCAO mice. PPF
increased the number of Nissl-positive cells. (J) Number of
degenerative neurons in tMCAO mice was observed by FJC staining
(magnification, ×20; scale bar, 100 μm). (K) FJC staining
indicated PPF decreased the number of degenerating neurons in the
ischemic penumbra of tMCAO mice. *P<0.05,
**P<0.01. PPF, Propofol; tMCAO, transient middle
cerebral artery occlusion; HE, hematoxylin and eosin; FJC,
Fluoro-Jade C; i.p., intraperitoneal injection.

Figure 6

PPF suppresses microglial activation
and neuronal injury in tMCAO mice. (A) Co-localization of Iba1 and
CD86 was detected by immunofluorescence. (magnification, ×40; scale
bar, 50 μm). (B) Immunofluorescence colocalization revealed
an increased proportion of CD86+ Iba1+ cells
in tMCAO mice, which was decreased by PPF treatment. ELISA
detection of TNF-α, IL-10, IFN-γ, and TGF-β levels in the ischemic
(C) core and (D) penumbra and (E) non-ischemic zone of brain
tissue. (F) Ischemic penumbra of tMCAO mice exhibited heightened
LDH levels. PPF reduced LDH levels. (G) ELISA revealed decreased
Cla, Occ and ZO-1 levels in tMCAO mice, while PPF elevated these
tight junction-associated protein levels. (H) Western blotting of
SYP-1, PSD-95, BDNF, GAP-43, and GAPDH (internal reference). (I)
Western blotting revealed decreased SYP-1 and PSD-95 levels, along
with increased BDNF and GAP-43 levels in tMCAO mice; these effects
were reversed by PPF. *P<0.05,
**P<0.01. PPF, Propofol; tMCAO, transient middle
cerebral artery occlusion; Iba, Ionized calcium binding adaptor
molecule; LDH, lactate dehydrogenase; Cla, Claudin; Occ, Occludin;
ZO-1, zona occludens 1; PSD, postsynaptic density protein; BDNF,
brain-derived neurotrophic factor; GAP, growth-associated protein;
SYP, synapsin.

Figure 7

PPF upregulates MFG-E8 to inhibit
activation of NF-κB/NLRP3 pathway. (A) Western blotting of MFG-E8,
p-NF-κB, NF-κB, NLRP3, ASC, cleaved-caspase-1, pro-caspase-1,
GSDMD-N, GSDMD, IL-18, IL-1β, and GAPDH (internal reference). (B-F)
Western blot analysis revealed decreased MFG-E8 (B) levels in tMCAO
mice, along with elevated p-NF-κB/NF-κB (C), NLRP3, ASC, IL-1β,
IL-18 (D), cleaved-caspase-1/pro-caspase-1 (E), GSDMD-N/GSDMD (F)
levels. PPF reduced these protein levels. (G) Western blotting
protein bands for MFG-E8 and GAPDH (internal reference). (H)
Western blotting detected the expression of MFG-E8 in the brain
tissue of mice after injection of si-MFG-E8/si-NC. (I) Western
blotting protein bands for MFG-E8, p-NF-κB, NF-κB, NLRP3, ASC,
cleaved-caspase-1, pro-caspase-1, GSDMD-N, GSDMD, IL-18, IL-1β, and
GAPDH (internal reference). (J-N) Western blot analysis revealed
that si-MFG-E8 diminished the therapeutic efficacy of PPF, leading
to deceased MFG-E8 level (J) and elevated p-NF-κB/NF-κB (K), NLRP3,
ASC, IL-1β, and IL-18 levels (L), and
cleaved-caspase-1/pro-caspase-1 (M) and GSDMD-N/GSDMD (N) levels in
tMCAO mice. *P<0.05, **P<0.01. PPF,
Propofol; tMCAO, transient middle cerebral artery occlusion;
MFG-E8, milk fat globule-EGF factor 8; p-, phosphorylated; ASC,
apoptosis-associated speck-like protein containing a CARD; ns, not
significant; GSDMD, gasdermin D; si, small interfering; NC,
negative control.

Figure 8

Mechanism of action of Propofol in
attenuating I/R neuronal injury. Propofol ameliorates neuronal
injury in cerebral I/R injury by inhibiting NF-κB/NLRP3
pathway-mediated focal death through upregulation of MFG-E8. I/R,
ischemia/reperfusion; LDH, lactate dehydrogenase; GSDMD, gasdermin
D; MFG-E8, milk fat globule-EGF factor 8; ASC, apoptosis-associated
speck-like protein containing a CARD.
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Copy and paste a formatted citation
Spandidos Publications style
Guo S, Zhen Y, Zhou G and Zhao Z: Propofol upregulates MFG‑E8 in BV2 cells to inhibit pyroptosis mediated by the NF‑&kappa;B/NLRP3 pathway, thereby ameliorating ischemic‑reperfusion neuronal injury. Int J Mol Med 57: 115, 2026.
APA
Guo, S., Zhen, Y., Zhou, G., & Zhao, Z. (2026). Propofol upregulates MFG‑E8 in BV2 cells to inhibit pyroptosis mediated by the NF‑&kappa;B/NLRP3 pathway, thereby ameliorating ischemic‑reperfusion neuronal injury. International Journal of Molecular Medicine, 57, 115. https://doi.org/10.3892/ijmm.2026.5786
MLA
Guo, S., Zhen, Y., Zhou, G., Zhao, Z."Propofol upregulates MFG‑E8 in BV2 cells to inhibit pyroptosis mediated by the NF‑&kappa;B/NLRP3 pathway, thereby ameliorating ischemic‑reperfusion neuronal injury". International Journal of Molecular Medicine 57.5 (2026): 115.
Chicago
Guo, S., Zhen, Y., Zhou, G., Zhao, Z."Propofol upregulates MFG‑E8 in BV2 cells to inhibit pyroptosis mediated by the NF‑&kappa;B/NLRP3 pathway, thereby ameliorating ischemic‑reperfusion neuronal injury". International Journal of Molecular Medicine 57, no. 5 (2026): 115. https://doi.org/10.3892/ijmm.2026.5786
Copy and paste a formatted citation
x
Spandidos Publications style
Guo S, Zhen Y, Zhou G and Zhao Z: Propofol upregulates MFG‑E8 in BV2 cells to inhibit pyroptosis mediated by the NF‑&kappa;B/NLRP3 pathway, thereby ameliorating ischemic‑reperfusion neuronal injury. Int J Mol Med 57: 115, 2026.
APA
Guo, S., Zhen, Y., Zhou, G., & Zhao, Z. (2026). Propofol upregulates MFG‑E8 in BV2 cells to inhibit pyroptosis mediated by the NF‑&kappa;B/NLRP3 pathway, thereby ameliorating ischemic‑reperfusion neuronal injury. International Journal of Molecular Medicine, 57, 115. https://doi.org/10.3892/ijmm.2026.5786
MLA
Guo, S., Zhen, Y., Zhou, G., Zhao, Z."Propofol upregulates MFG‑E8 in BV2 cells to inhibit pyroptosis mediated by the NF‑&kappa;B/NLRP3 pathway, thereby ameliorating ischemic‑reperfusion neuronal injury". International Journal of Molecular Medicine 57.5 (2026): 115.
Chicago
Guo, S., Zhen, Y., Zhou, G., Zhao, Z."Propofol upregulates MFG‑E8 in BV2 cells to inhibit pyroptosis mediated by the NF‑&kappa;B/NLRP3 pathway, thereby ameliorating ischemic‑reperfusion neuronal injury". International Journal of Molecular Medicine 57, no. 5 (2026): 115. https://doi.org/10.3892/ijmm.2026.5786
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