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. 2021 Jan 25:15:277-287.
doi: 10.2147/DDDT.S267856. eCollection 2021.

Physcion Protects Rats Against Cerebral Ischemia-Reperfusion Injury via Inhibition of TLR4/NF-kB Signaling Pathway

Xiaobo Dong #  1 , Lei Wang #  1 , Guangrong Song  1 , Xu Cai  1 , Wenxin Wang  1 , Jiaqi Chen  1 , Gesheng Wang  1
Affiliations

Physcion Protects Rats Against Cerebral Ischemia-Reperfusion Injury via Inhibition of TLR4/NF-kB Signaling Pathway

Xiaobo Dong et al. Drug Des Devel Ther. .

Abstract

Background: Ischemic stroke (IS) is characterized by the rapid loss of brain function due to ischemia. Physcion has been found to have a neuroprotective effect against cerebral ischemia-reperfusion (I/R) injury. However, the mechanism by which physcion regulates cerebral I/R injury remains largely unknown.

Methods: An oxygen-glucose deprivation/reperfusion (OGD/R) model in SH-SY5Y cells and a rat cerebral ischemia-reperfusion (I/R) model were established, respectively. CCK-8 and flow cytometry assays were used to detect the viability and apoptosis of SH-SY5Y cells. Moreover, enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of SOD, MDA, GSH-Px, TNF-α, IL-1β, IL-6 and IL-10 in the supernatant of SH-SY5Y cells. Meanwhile, Western blot assay was used to detect the expressions of TLR4, p-p65 and p-IκB in SH-SY5Y cells and I/R rats.

Results: In this study, physcion treatment significantly rescued OGD/R-induced neuronal injury. In addition, physcion decreased inflammatory response in SH-SY5Y cells after OGD/R insult, as shown by the decreased levels of the pro-inflammatory factors TNF-α, IL-1β, IL-6 and IL-10. Moreover, physcion attenuated the oxidative stress in OGD/R-treated SY-SY5Y cells, as evidenced by the increased SOD and GSH levels and the decreased ROS and MDA levels. Meanwhile, physcion significantly reduced cerebral infarction, attenuated neuronal injury and apoptosis in I/R rats. Furthermore, physcion markedly decreased the expressions of TLR4, p-NF-κB p65 and p-IκB in the brain tissues of rats subjected to I/R and in SH-SY5Y cells exposed to OGD/R.

Conclusion: In conclusion, our study indicated that physcion protected neuron cells against I/R injury in vitro and in vivo by inhibition of the TLR4/NF-kB pathway; thus, physcion might serve as a promising therapeutic candidate for IS.

Keywords: NF-κB; ischemia-reperfusion injury; physcion; stroke.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Physcion protects cell against OGD/R-induced neuronal injury. (A) SH-SY5Y cells were treated with 0, 10, 20, 40 or 80 μM physcion for 24 h. Cell viability was determined using CCK-8 assay. (B) SH-SY5Y cells were exposed to OGD for 4 h, and then reoxygenated in the presence of 10, 20, or 40 μM physcion for 24 h. Cell viability was determined using CCK-8 assay. (C) Cell cytotoxicity was determined using LDH release assay. (D and E) Apoptotic cells were measured by flow cytometry. **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with OGD/R group.
Figure 2
Figure 2
Physcion attenuates OGD/R-induced oxidative stress in SH-SY5Y cells. SH-SY5Y cells were exposed to OGD for 4 h, and then reoxygenated in the presence of 40 μM physcion for 24 h. (A and B) Intracellular ROS generation was assessed by flow cytometry. (C–E) Levels of SOD, MDA and GSH-Px in cells were detected with ELISA. **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with OGD/R group.
Figure 3
Figure 3
Physcion suppresses OGD/R-induced inflammatory response in SH-SY5Y cells. SH-SY5Y cells were exposed to OGD for 4 h, and then reoxygenated in the presence of 40 μM physcion for 24 h. (A–E) Levels of IL-1β, TNF-α, IL-10, IL-6 and MCP-1 in cells were detected with ELISA. **P<0.01 compared with control group; ##P<0.01 compared with OGD/R group.
Figure 4
Figure 4
Physcion attenuates OGD/R-induced injury in SH-SY5Y cells via inhibiting the TLR4/p65 pathway. SH-SY5Y cells were exposed to OGD for 4 h, and then reoxygenated in the presence of 40 μM Oroxylin A for 24 h. (A) Expression levels of TLR4, p-p65, p-IκB in SH-SY5Y cells were detected with Western blotting. (B–D) The relative expressions of TLR4, p-p65, p-IκB in cells were quantified via normalization to β-actin, p65 and IκB. **P<0.01 compared with control group; ##P<0.01 compared with OGD/R group.
Figure 5
Figure 5
Physcion protects rats against brain damage after I/R. (A and B) TTC staining was applied to assess infarct area (magenta: healthy tissue; white: damaged tissue). (C) Cerebral water content was calculated. (D) Neurobehavioral tests were performed at 6 h, 1, 3, 5 or 7 days after MCAO. Neurological severity scores (NSS). (E) HE staining assay was performed to detect brain tissue injury in I/R rats (magnification x 200). (F) TUNEL assay was performed to detect cell apoptosis in I/R rats challenged with MCAO (magnification x 400). **P<0.01 compared with sham group; #P<0.05, ##P<0.01 compared with I/R group.
Figure 6
Figure 6
Physcion protects rats against brain damage after I/R via inhibiting the TLR4/p65 pathway. (A) Expression levels of TLR4, p-p65, p-IκB in the whole brain tissues of I/R rats were detected with Western blotting. (B–D) The relative expressions of TLR4, p-p65, p-IκB in the whole brain tissues of I/R rats were quantified via normalization to β-actin, p65 and IκB. **P<0.01 compared with sham group; ##P<0.01 compared with I/R group.
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Grants and funding

This study received funding from the State Administration of Traditional Chinese Medicine of the Special Scientific Research on the Business Construction of National TCM Clinical Base (No. JDZX2015043) and Key Research Projects of Beijing University of Chinese Medicine in 2020 (No. 2020-JYB-ZDGG-129).

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