Ginseng Rh2 protects endometrial cells from oxygen glucose deprivation/re-oxygenation

Abstract

In this study, oxygen glucose deprivation/re-oxygenation (OGDR) was applied to cultured endometrial cells to mimic ischemic-reperfusion injuries. We also tested the potential effect of Ginseng Rh2 (GRh2) against the process. In established T-HESC human endometrial cells and primary murine endometrial cells, GRh2 largely inhibited OGDR-induced viability reduction and cell death. Remarkably, OGDR induced programmed necrosis in the endometrial cells, evidenced by cyclophilin D-p53-adenine nucleotide translocator 1 (ANT-1) mitochondrial association, mitochondrial depolarization, reactive oxygen species production, and lactate dehydrogenase release. Notably, such effects by OGDR were largely attenuated with co-treatment of GRh2. Further, cyclophilin D inhibition or knockdown also protected endometrial cells from OGDR. On the other hand, forced over-expression of cyclophilin D facilitated OGDR-induced T-HESC cell necrosis, which was dramatically inhibited by GRh2. Together, GRh2 protects endometrial cells from OGDR possibly via inhibiting CypD-dependent programmed necrosis pathway.

Keywords: Ginseng Rh2; cyclophilin; endometrial cells; oxygen glucose deprivation/re-oxygenation; programmed necrosis.

Figure 1. GRh2 protects endometrial cells from OGDR

The T-HESC human endometrial cells (A and B) or the primary murine endometrial cells (C and D) were treated with applied concentration (1-25 μM) of GRh2, together with/out OGD exposure for 4 hours, followed by 24 hours of re-oxygenation (“OGDR”), cell survival was tested by CCK-8 assay (A and C); cell death was examined by the trypan blue staining assay (B and D). “OGDR” stands for OGD/re-oxygenation (same for all figures). “Ctrl” stands for untreated control cells (same for all figures). For the OGDR experiments, endometrial cells were always pre-treated with applied concentration of GRh2 for 30 min before OGD (same for all figures). Bars stands for mean ± standard deviation (SD, n=5). ^* p<0.05 vs. “Ctrl”. ^# p<0.05 vs. cells with “OGDR” only treatment (no GRh2). Each experiment was repeated four times with similar results obtained.

Figure 2. OGDR fails to induce endometrial cell apoptosis

The T-HESC human endometrial cells (A-F) or the primary murine endometrial cells (G) were treated with GRh2 (10 μM), with/out OGDR exposure, after applied time, cell apoptosis was tested by the assays mentioned in the text (A-E). LDH release in the conditional medium was tested as the indicator of cell necrosis (F and G). For testing cell apoptosis, cell permeable short-chain C6 ceramide (“C6”, 20 μM, 24 hours) was added as the positive control (A-E). Bars stands for mean ± standard deviation (SD, n=5). ^* p<0.05 vs. “Ctrl”. ^# p<0.05 vs. cells with “OGDR” only treatment (no GRh2). Each experiment was repeated four times with similar results obtained.

Figure 3. GRh2 prevents OGDR-induced programmed necrosis in endometrial cells

T-HESC human endometrial cells were treated with GRh2 (10 μM), together with/out OGDR exposure, after applied time, mitochondrial CypD-p53-ANT-1 association (“Mito-IP”, A, “Input” showed expression of the proteins in total cell lysates), mitochondrial depolarization (JC-1 intensity OD, B), ROS production (C), lipid peroxidation (D) and cytochrome C release (E, testing cytosol proteins) were tested by the assays mentioned in the text. For the Mito-IP assay, CypD-bound p53 and CypD-bound ANT-1 were quantified (A, the right panel). For the cytochrome C release assay, relative cytosol cytochrome C level (vs. Tubulin) was also quantified (E, the lower panel). Bars stands for mean ± standard deviation (SD, n=5). ^* p<0.05 vs. “Ctrl”. ^# p<0.05 vs. cells with “OGDR” only treatment (no GRh2). Each experiment was repeated three times with similar results obtained.

Figure 4. Inhibition of CypD prevents OGDR-induced endometrial cell programmed necrosis

T-HESC cells, expressing the scramble non-sense control shRNA (“sh-C”) or CypD-targeting shRNA (“shCypD”) were treated with/out OGD for 4 hours (“sh-C” cells were also co-treated with cyclosporin A “CsA,10 μM”), followed re-oxygenation (“OGDR”) for applied time, expressions of CypD and other listed genes were tested by qRT-PCR assay (A) and Western blotting assay (B); cell survival and necrosis were tested by CCK-8 assay (C) and LDH release assay (D), respectively. The primary murine endometrial cells were treated with 10 μM of CsA, together with/out OGDR for 24 hours, cell survival (E) and necrosis (F) were tested. Bars stands for mean ± standard deviation (SD, n=5). “DMSO” stands for 0.1% of DMSO. ^* p<0.05 vs. “Ctrl”. ^# p<0.05 vs. cells with “OGDR” only treatment (no GRh2). Each experiment was repeated three times with similar results obtained.

Figure 5. Forced over-expression of CypD facilitates OGDR-induced endometrial cell death, inhibited by GRh2

T-HESC cells, expressing the empty vector (pSuper-puro-Flag, “Vector”) or CypD-Flag vector (“CypD-Flag”), were treated with GRh2 (10 μM), together with/out OGDR exposure for 24 hours, CypD mRNA and protein expressions were tested by qRT-PCR assay (A) and Western blotting assay (B), respectively; cell survival and necrosis were tested by CCK-8 assay (C) and LDH release assay (D), respectively. Bars stands for mean ± standard deviation (SD, n=5). ^* p<0.05 vs. “Ctrl”. ^# p<0.05 vs. cells with “OGDR” only treatment (no GRh2). Each experiment was repeated three times with similar results obtained.