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

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.

Ginsenosides are the primary active constituents in the root of Ginseng [20,21]. Ginsenoside Rh2 (GRh2) is a converted ginsenoside, which has shown various pharmaceutics activities [20,21]. In the current study, we show that GRh2 protects endometrial cells from OGDR possibly via inhibiting programmed necrosis pathway.
The potential effect of GRh2 in the primary endometrial cells was also tested. As demonstrated, treatment the primary murine endometrial cells with the same OGDR procedure (4 hours of ODG plus 24 hours of re-oxygenation) induced profound viability (CCK-8 OD) reduction ( Figure 1C) and cell death ( Figure 1D). Co-treatment with 10 μM of GRh2 again significantly attenuated OGDR-induced cytotoxicity in the primary murine endometrial cells ( Figure 1C and 1D). GRh2 alone was non-cytotoxic to the primary endometrial cells ( Figure  1C and 1D). Together, these results suggest that GRh2 protects endometrial cells from OGDR.

OGDR fails to induce endometrial cell apoptosis
Cell apoptosis is a main reason of cell death in response to different stimuli. We thus tested possible apoptosis induction in OGDR-treated endometrial cells.
Cell necrosis is another form of cell death [15]. Lactate dehydrogenase (LDH) release to the medium is often detected as a marker of cell necrosis in vitro. In the current study, we showed that medium LDH content was significantly increased following OGDR exposure in T-HESC endometrial cells ( Figure 2F). Remarkably, such effect was largely inhibited by co-treatment of GRh2 ( Figure 2F). These results imply that GRh2 might protect T-HESC cells from OGDR via inhibiting cell necrosis, but not apoptosis. In the primary murine endometrial cells, GRh2 (10 μM) similarly inhibited OGDR-induced cell necrosis (LDH release, Figure 2G).

Inhibition of CypD prevents OGDR-induced endometrial cell programmed necrosis
To further confirm that programmed necrosis pathway activation is required for OGDR-induced endometrial cell death, pharmacologic and shRNA methods were employed to inhibit CypD. Cyclosporin A (CsA) is a known CypD inhibitor, which is shown to shut down the programmed necrosis pathway [11,12,31,32]. Additionally, two shRNAs, against distinct and non-overlapping sequence of CypD (a gift from Dr. Guo [11]), were applied. CypD mRNA ( Figure 4A) and protein ( Figure 4B) expressions were largely downregulated in the stable T-HESC cells with the CypD shRNA. CsA or OGDR alone didn't change CypD expression ( Figure   4A). As demonstrated, inhibition of CypD by CsA or the two targeted-shRNAs largely attenuated OGDRinduced T-HESC cell viability (CCK-8 OD) reduction ( Figure 4C) and cell necrosis (LDH medium release, Figure 4D). Thus, inhibition of CypD activation (by CsA) or expression (by targeted-shRNA) could efficiently alleviate OGDR-induced T-HESC cell death ( Figure 4C and 4D). These evidences further suggest that CypDdependent necrosis pathway mediates cell death by OGDR. In the primary murine endometrial cells, the CypD inhibitor CsA similarly alleviated cell death (CCK-8 OD reduction, Figure 4E) and necrosis (LDH release, Figure  4F) by OGDR. It should be noted that CypD inhibition or knockdown alone failed to change endometrial cell survival and death ( Figure 4A-4F).

Forced over-expression of CypD facilitates OGDR-induced endometrial cell death, inhibited by GRh2
Based on the above results, we propose that CypDdependent programmed necrosis pathway activation mediates OGDR-induced endometrial cell death. We , 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. therefore speculated that forced over-expression of CypD might possibly facilitate OGDR-induced endometrial cell death. To test this hypothesis, a CypD-expression vector (a gift from Dr. Fang [13]) was applied. The exogenous CypD construct was tagged with Flag, and was transfected to T-HESC endometrial cells. CypD mRNA expression was significantly increased in cells with the construct (Figure 5A), which was not changed by GRh2 nor OGDR ( Figure 5A). Western blotting assay results in Figure 5B further confirmed the expression of exogenous CypD (Flag-tagged) in the stable cells.
Significantly, OGDR-induced viability reduction ( Figure  5C) and cell necrosis ( Figure 5D) were significantly boosted in T-HESC cells with the exogenous CypD. These results demonstrated that over-expression of CypD indeed facilitated OGDR-induced endometrial cell death. Notably, co-treatment with GRh2 potently inhibited OGDR-induced death even in the CypD-overexpressed T-HESC cells ( Figure 5C and 5D). These results again suggest that GRh2-induced endometrial cell protection is possibly through inhibiting CypDdependent programmed necrosis pathway. 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. www.impactjournals.com/oncotarget DISCUSSION p53 is a key molecule to mediate cell apoptosis [33][34][35][36][37]. Recent studies, however, have implied that p53 is also required for the induction of cell necrosis. Various stimuli and stresses, i.e. hypoxia, calcium overload, and oxidative stresses, could trigger p53 translocation to mitochondria, where it associates with CypD [27]. Mitochondrial Cyp-D-p53 complexation is proven to be required for the mitochondrial depolarization [27], and more importantly, subsequent cell necrosis (programmed necrosis). Inhibition of the CypD-p53 complex, genetically or pharmacologically, could efficiently protect cells from the stresses [27].
In the current study, we proposed that OGDR also induced programmed necrosis in endometrial cells, which was evidenced by mitochondrial CypD-p53-ANT-1 association (a key step to initiate necrosis [11, 12, 17,

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. www.impactjournals.com/oncotarget 26]), mitochondrial depolarization, ROS production, and LDH medium release (the end point). On the other hand, inhibition of programmed necrosis pathway, by CypD shRNA or its inhibitor CsA, significantly inhibited OGDR-induced endometrial cell death. Notably, results of various apoptosis assays indicated that OGDR failed to induce significant apoptosis activation in endometrial cells. Therefore, CypD-dependent programmed necrosis pathway could be an important therapeutic target for the treatment of endometrial cell OGDR/ischemia-reperfusion injuries.
Although GRh2 has displayed many different functions in various cell types, this is, as far as we know, the first study to test its potential activity in endometrial cells. Here, we showed that GRh2 efficiently protected T-HESC human endometrial cells and primary murine endometrial cells from OGDR. At the molecular level, we propose that inhibition of the programmed necrosis pathway could be the primary mechanism of its cytoprotection against OGDR. Although the underlying mechanisms need further characterizations, our results imply that it would be interesting to further test GRh2's activity against endometrial cell ischemia-reperfusion injuries.

Chemical and reagents
GRh2, the CypD inhibitor cyclosporine A (CsA), and puromycin were purchased from Sigma-Aldrich (Shanghai, China). The two non-overlapping lentiviral CypD shRNAs ("-1/-2") as well as the scramble control non-sense lentiviral shRNA were provided by Dr. Guo [11]. All the reagents for cell culture were purchased from Gibco (Suzhou, China). GRh2 was always freshly dissolved in warm phosphate buffer solution (PBS) before the experimental usage. The antibodies utilized in this study were provided by Santa Cruz Biotechnology (Santa Cruz, CA) and Cell Signaling Tech (Suzhou, China). The short-chain C6 ceramide was purchased from Avanti Polar Lipids (Alabaster, Alabama).

Culture T-HESC human endometrial cells
The immortalized human endometrial cell line, T-HESC [22], was purchased from the Cell Bank of Shanghai Institute of Biological Science of CAS (Shanghai, China). The T-HESC cells were cultured in DMEM-F12 medium with 10% fetal bovine serum (FBS), and 4 mM L-glutamine, 0.25% HEPES plus necessary antibiotics, at 37°C in a humidified atmosphere of 5% CO 2 [22].

The primary culture of murine endometrial (stromal) cells
Female, [8][9][10][11][12] week-old C57BL/6J mice were purchased from the Experimental Animal Center of Soochow University (Suzhou, China). Animals were housed in filter-top cages under conventional conditions. The detailed protocol was described in detail in a previous study [38]. Three days prior to hysterectomy, mice (n = 5/culture) were injected s.c. with 100 ng of estrogen E2 solution to synchronize the cycle. The acquired primary mouse uterine tissues were then incubated with 0.05% trypsin-EDTA supplemented with 1 mg/mL collagenase (Sigma-Aldrich) for 30 min at 37°C. Next, the uterine tissues were transferred to DMEM/Hams F-12 nutrient plus 10% FBS. After 5 min, tissues were transferred into 3 mL of ice-cold HBSS and vortexed. The resulting epithelial cells were abandoned using gravity sedimentation, and the step was repeated three times. Afterwards, the primary endometrial stromal cells were pelleted and resuspended in the complete DMEM medium with 10% FBS (Gibco, Belgium), 0.5 mg/mL fungizone and 100 mg/mL gentamicin. The animal protocol was approved by the Ethics Review Board of Soochow University (Suzhou, China).

Cell viability assay
The viability of endometrial cells was tested by cell counting kit-8 (CCK-8) kit (Dojindo Laboratories, Kumamoto, Japan), according to the standard procedure. The CCK-8 optic density (OD) at 450 nm was recorded.

Trypan blue staining of "dead" cells
Dead cells were positively stained with trypan blue dye (Sigma), and the percentage (%) of trypan blue-stained cells was recorded (using an automatic cell counter), indicating cell death ratio.

Lactate dehydrogenase (LDH) assay
The release of LDH from the intact cells to the conditional medium is a characteristic marker of cell necrosis in vitro [39]. In this study, a two-step LDH detection kit (Promega, Shanghai, China) was employed to test LDH level in the medium, and its level was always normalized to total LDH (combination of medium LDH and cellular LDH).

OGD/re-oxygenation (OGDR)
The OGDR procedure was described in a previous study [40]. Briefly, endometrial cells were first placed into an airtight chamber and equilibrated for 10 min with a continuous flux of gas (95% N 2 /5% CO 2 ). The chamber was sealed and placed in an incubator for 4 hours (mimic oxygen glucose deprivation or OGD). Afterwards, the endometrial cells returned back to the complete medium and re-oxygenated. Control cells were placed in normoxygenated complete medium. www.impactjournals.com/oncotarget

Western blotting assay
Following the indicated treatment, the endometrial cells were incubated with RIPA lysis buffer (Biyuntian, Wuxi, China). The quantified protein lysate samples (40 μg per treatment of each lane) were separated by the 10-12% SDS-PAGE gels, and were transferred to the polyvinylidene difluoride (PVDF) membrane [41]. The detailed protocol of Western blotting assay and data quantification were described previously [42,43].

Caspase-3 activity assay
For each treatment, 20 μg of cytosolic extracts were incubated with the caspase assay buffer [12] together with the caspase-3 substrate [12] for 2 hours at the room temperature, the release of 7-amido-4-(trifluoromethyl) coumarin (AFC) was quantified, using a Fluoroskan system set [12]. The AFC's OD value (at 405 nm) was recorded to reflect caspase-3 activity.

Hoechst-33342 nuclei staining of apoptotic cells
Cells were stained with Hoechst-33342 (Sigma). Nonapoptotic nuclei were with faint delicate chromatin staining, but nuclei with intensified Hoechst-33342 condensation/ brightness (early apoptotic cells) or fragmentation (late apoptotic cells) were labeled as apoptotic cells. Apoptotic nuclei percentage was calculated, from at least 300 cells of 5 random views of each treatment.

Other apoptosis assays
The Annexin V FACS assay of cell apoptosis and TUNEL nuclei staining assay of apoptosis were described in detail in the previous studies [44]. Annexin V ratio was sorted by Becton-Dickinson FACScan (Shanghai, China). TUNEL ratio (vs. total number of cells) was calculated, from at least 300 cells of 5 random views for each treatment.

Mitochondrial immunoprecipitation (Mito-IP)
After the applied treatment, T-HESC cells were harvested and homogenized by the buffer of 250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, and 1 mM dithiothreitol. After centrifugation, the supernatants were collected as the cytosolic fractions. The resulting pellets were then resuspended in the above buffer plus 10 μL NP-40, which were the mitochondrial fraction lysates. The quantified mitochondrial lysates (400 μg per sample) were precleared and incubated with anti-CypD antibody [27,45]. The mitochondrial complexes were then captured by the protein G-Sepharose beads (Sigma). CypD-p53-ANT-1 association was then tested by Western blotting assay.

Mitochondrial depolarization assay
JC-1 fluorescent intensity change is often tested to reflect the mitochondrial membrane potential change [46]. With mitochondrial depolarization ("ΔΨ"), the red JC1 aggregates shall form the green monomers [47]. Testing mitochondrial depolarization by the JC-1 protocol was discussed in previous studies. Briefly, T-HESC cells were incubated with JC-1 (10 μg/mL, Invitrogen, Shanghai, China) for 10 min at the room temperature under the dark. JC-1 green fluorescence OD, reflecting mitochondrial depolarization, was examined immediately on a fluorescence spectrofluorometer at 530 nm (Titertek Fluoroscan, Germany).

Lipid peroxidation assay
As previously described [50], cellular lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBAR) assay [51]. Briefly, 20 μg lysate proteins of each treatment were first mixed with 20% of acetic acid and thiobarbituric acid solution. After heating, the mixture was centrifuged, and the red pigment dye in the supernatant was examined by the microplate reader [51]. TBAR activity was expressed as nM of malondialdehyde per mg protein.

qRT-PCR
After treatment, cellular RNA was extracted by the RNeasy Mini Kit (Qiagen, Shanghai, China). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA via a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Shanghai, China). The ABI Prism 7600 Fast Real-Time PCR system was utilized to perform the quantitative real time-PCR (qRT-PCR) assay. For each assay, melt curve analysis was performed to calculate product melting temperature. Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) mRNA was tested as the reference gene, and the 2 −ΔΔCt method was applied to quantify CypD mRNA expression change. The mRNA primers of human CypD and GAPDH were described previously [52]. www.impactjournals.com/oncotarget CypD shRNA T-HESC cells were cultured at 50% confluence in low-serum (1%) medium in six-well tissue culture plate. The CypD shRNA lentivirus (10 μL/mL medium per well) or the scramble control shRNA lentivirus (10 μL/mL medium per well) was added to T-HESC cells overnight. Stable clones were selected with puromycin (1 μg/mL) starting at 24 hours after infection, for a total of 8 days. All the resulting stable cells were then assayed for knockdown of CypD via qRT-PCR assay and Western blotting assy.

CypD over-expression
The CypD pSuper-puro-Flag vector and the empty vector were provided by Dr. Fang [13]. The CypD construct or the vector was transfected through Lipofectamine 2000 reagent (Invitrogen, Suzhou, China) to the cultured T-HESC cells. After 24 hours, cells were selected by puromycin (1.0 μg/mL) for another 8 days. CypD expression in the resulting stable cells was verified by both qRT-PCR assay and Western blotting assy.

Statistical analysis
All data were presented as mean ± standard deviation (SD). Repeated-measures analysis of variance (RMANOVA) followed by Dunnett's post hoc test for multiple comparisons (SPSS 16.0) were applied to evaluate statistical significance of observed differences.

CONCLUSION
Postpartum hemorrhage remains a prominent cause of maternal morbidity and mortality, which could possibly be managed by using a stepwise progressive approach [1][2][3]. Studies have demonstrated that manual and pharmacologic interventions are first-line treatments [1][2][3]. Our results provided new mechanistic insights (programmed necrosis) and related intervention strategies (GRh2) of ischemia-reperfusion-related endometrial cell injuries.

Author contributions
All listed authors carried out the experiments, participated in the design of the study and performed the statistical analysis, conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.