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. 2023 Oct 16;14(1):20220314.
doi: 10.1515/tnsci-2022-0314. eCollection 2023 Jan 1.

CST3 alleviates bilirubin-induced neurocytes' damage by promoting autophagy

Zhenkun Li  1 , Yating Du  2
Affiliations

CST3 alleviates bilirubin-induced neurocytes' damage by promoting autophagy

Zhenkun Li et al. Transl Neurosci. .

Abstract

High concentrations of unconjugated bilirubin (UCB) have toxic effects. The aim of our study was to find a way to elevate UCB tolerance or inhibit its toxicity in neurocytes. It has been reported that cystatin C (CST3) concentrations have a significant positive correlation with total bilirubin (TB) levels and a negative correlation with albumin levels. In addition, CST3 can directly bind UCB, decrease human umbilical vein endothelial cells' permeability, improve blood-brain barrier integrity after ischemic brain injury in mice, and induce autophagy. We hypothesized that CST3 could increase the solubility of UCB, decrease permeability of neurocytes, induce autophagy of neurocytes, and alleviate bilirubin-induced damage. To verify our hypothesis, we measured TB and conjugated bilirubin levels, and the permeability and autophagy of neurocytes treated with UCB and CST3. Our findings suggest that CST3 can protect against UCB-induced damage in neurocytes and that autophagy played an important role in this process.

Keywords: autophagy; cystatin C; hyperbilirubinemia; unconjugated bilirubin.

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Conflict of interest statement

Conflict of interest: Authors state no conflict of interest

Figures

Figure 1
Figure 1
CST3 alleviates injury to neurocytes in the high-dose UCB group: (a) CST3 mRNA expression levels, (b) CST3 protein expression levels, (c) viability of HT22 cells treated with UCB and CST3, (d) serum bilirubin contents of hyperbilirubinemic mice, (e) frequency swimming across the platform, (f) mean distance to platform, and (g) Masson staining in the brains of hyperbilirubinemic mice treated with CST3. CTL: control group; HBR: high-dose UCB group; DB: direct bilirubin; IB: indirect bilirubin. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
Figure 2
Figure 2
Effects of CST3 on UCB solubility in cell-free systems and permeability in HT22 cells: (a) UCB content in CTL and CST3 groups, (b) macromolecule permeability of HT22 cells of HBR and HBR + CST3 group, (c) UCB permeability in HT22 cells of HBR and HBR + CST3 groups, (d) expression level of UGT1A1, and (e) activity of cytochrome P450. CST3: adding CST3 protein; CST3+: CST3 overexpression; CST3−: CST3 knockdown. *p ≤ 0.05.
Figure 3
Figure 3
CST3 protects HT22 cells from UCB damage via promoting autophagy: (a) CST3 promotes HT22 cells autophagy, (b) mTOR, (c) pmTOR, (d) Beclin1, (e) LC3II/LC3I, (f) viability of HT22 cells treated with CST3 and autophagy inhibitor Bafilomycin A1, and (g) viability of HT22 cells treated with CST3 and autophagy activator rapamycin. HBR: high-dose UCB group; +: adding or overexpression; -: not adding; --: knockdown. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
Figure 4
Figure 4
CST3 alleviates UCB-induced damage to neurocytes by promoting autophagy. Arrows: autophagosomes; triangle: mitochondria.
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