The effect of function in mesenchymal stem cells after bilirubin stimulation

doi:10.21203/rs.3.rs-1750223/v1

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The effect of function in mesenchymal stem cells after bilirubin stimulation

https://doi.org/10.21203/rs.3.rs-1750223/v1

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Objective

Neonatal bilirubin encephalopathy is a serious complication of neonatal hyperbilirubinemia. There is no good treatment for its. At present, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) therapy has been widely concerned, but its effect on bilirubin encephalopathy is not clear. Therefore, this study intends to explore the stimulation of hUC-MSCs by bilirubin in vitro, and provide theoretical basis for the further treatment of bilirubin encephalopathy with MSCs.

Methods

hUC-MSCs were added in control group (0mg/dL group), and bilirubin concentration group(5 mg/dL, 10 mg/dL and 15 mg/dL) for 12h and 24h. The survival rate after bilirubin stimulation was detected by CCK-8, the adipogenic and osteogenic differentiation was induced to observe the change of differentiation ability, IL-6, TSG-6 level was measured by ELISA, Western boltting measured Bax Bcl-2 caspase 3 protein level.

Results

The number of cells decreased and the morphology did not change significantly. The cell survival rate of each group was statistically significant 12h after bilirubin stimulation (P < 0.001). After induction, there was no significant difference in staining results among all groups. This indicated that bilirubin had no significant effect on the osteogenic adipogenic differentiation of hUC-MSCs.The level of IL-6, TSG-6, Bax, Bcl-2 and capcase-3 increased significantly after bilirubin stimulated MSCs compared with the control group (P < 0.05).

Conclusion

Bilirubin have toxic effects for hUC-MSCs, with the increase of bilirubin concentration will affect the proliferation secretion and apoptosis, but it had no obvious effect on their differentiation ability.

Neonate

Bilirubin encephalopathy

Mesenchymal stem cells

Paracrine

Jaundice is one of the most common manifestation in neonates. About 60% of full-term and 80% of preterm infants develop jaundice in the first week of life. Pathological jaundice can cause hyperbilirubinemia, especially when it occurs simultaneously with infection, ischemia, hypoxia and acidosis, it may develop into bilirubin encephalopathy[1]. The main causes of bilirubin encephalopathy are the function of blood-brain barrier is damaged, in the meantime with the binding capacity of bilirubin decreasing, unbound bilirubin (UB) increasing[2, 3]. At present, bilirubin encephalopathy is mainly treated by drugs, phototherap and exchange transfusion. Despite aggressive treatment, neonatal bilirubin encephalopathy still has a variety of sequelae covers a wide spectrum of disorders characterized by varying degrees of auditory and motor dysfunction that affect the child's life[4].

In recent years, MSCs research has developed rapidly. It can be used to treat a variety of neonatal diseases and their complications, including bronchopulmonary dysplasia (BPD), hypoxic ischemic encephalopathy (HIE) and necrotizing enterocolitis (NEC)[5, 6]. Many studies have shown that MSCs can be isolated from bone marrow, adipose, bones, muscles, lungs, liver, and perinatal amniotic fluid, umbilical cord, umbilical cord blood, etc[7]. Perinatal umbilical cord-derived MSCs have been noticed by researchers[8]. Nowadays, MSCs not only have a strong differentiation effect, but also have been applied in the treatment of inflammation through the paracrine mechanism. It has been demonstrated that MSCs treatment of BPD or HIE mainly produces anti-inflammatory and anti-apoptotic effects through paracrine mechanisms[9–11]. MSCs have been shown in studies to secrete and relieve symptoms of disease by growth factors and cytokines including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), IL-6, IL-10, TGF-β, TSG-6, PGE-2 [12–14]. Now MSCs have been transplanted into rat models with severe intraventricular hemorrhage to improve brain injury, and this study showed that BDNF significantly increased[15–17]. This proves that MSCs have a significant therapeutic effect in the prognosis of brain injury. For example, in sepsis mouse model, inflammation-related genes were down-regulated and immune genes up-regulated after MSCs transplantation [18]. It was observed that MSCs could secret TSG-6, thereby alleviating inflammation and pancreatic damage in severe acute pancreatitis (SAP) rats[19]. It can be seen that the paracrine function of MSCs have been widely applied in the treatment of various systemic diseases. There's a lot of research on nervous system diseases that we're interested in such as HIE, traumatic brain injury (TBI)[20], cerebral palsy(CP)[21], etc. It can be found that the MSCs therapy can promote the recovery of neurological function, reduce the submit of brain injury, and alleviate the sequelae of nervous system such as motor disorders. However, there are few literatures on the treatment of bilirubin encephalopathy with MSCs. Some experiments have used adipose derived MSCs to treat animal models of bilirubin encephalopathy[22]. But the current MSCs therapy technology is still immature. Ours experiment observes the function changes of MSCs at different bilirubin concentration, so as to provide relevant evidence for the treatment of bilirubin encephalopathy by MSCs.

1.1 MSCs Culture

The hUC-MSCs were donated by Chongqing Engineering Research Center of Stem Cell Therapy and cultured with Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS;Gibco), penicillin, and streptomycin(Gibco) under 37℃,5% CO₂. The cells were passaged every 3-5 days.

1.2 Cell Viability by Cell Counting Kit-8(CCK-8)

Bilirubin (sigma,USA) was dissolved in dimethyl sulphoxide (DMSO), and stored away from light. The hUC-MSCs were plated at 1×10⁵ cells/well on 96-well plates. After 24h of culture in DMEM, differentconcentrations of bilirubin (0, 5 mg/dL, 10 mg/dL and 15 mg/dL) were added into the medium. After 12h and 24h, the medium was discarded, and 100 μl of 10% CCK-8 was added to each well. After 4h, the absorbance at 450 nm was measured with a microplate reader, and the cell survival rate was calculated.

1.3 MSCs Adipogenic Differentiation

The MSCs adipogenic differentmedium A and B were prepared. The 6-well plate was coated with gelatin, and the MSCs were inoculated into it at the cell density of 2×10⁴ cells/cm².12h later, bilirubin intervention as shown above, the medium was sucked away, washed with PBS for 3 times, and added the solution A. After 3 days of induction, solution A was aspirated and 2 mL solution B was added. After 1 day, solution A was changed back for induction. Alternate solution A and solution B, and observe cell status daily until sufficient lipid droplets of appropriate size are present to begin staining by oil red O solution.

1.4 MSCs Osteogenic Differentiation

The MSCs osteogenic differentmedium were prepared. Same as above for cell density. After 12h, wash the board same as above, then added the osteogenic differentmedium into the 6-well plate. It replaced every three days. After 28 days of induction, the cells were stained with alizarin red according to their morphological changes and growth.

1.5 Cytokines by ELISA

The hUC-MSCs were plated at 1×10⁶ cells/well on 6-well plates. Supernatant from cells after bilirubin intervention is used from ELISA. Follow the instructions of the kit to detect IL-6、TSG-6. The absorbance value was measured same as above, and the concentration of the corresponding factor was calculated by drawing a standard curve.

1.6 Western boltting

After bilirubin stimulation, the cells were lysed by radioimmunoprecipitation assay (RIPA), the upper liquid was absorbed as protein. Protein concentrations were measured by a bicinchoninic acid (BCA) protein assay kit and denatured in the sample holes of 20 μl SDS-PAGE for every 80 μl protein added. The same amount of protein(30ug) was added in each lane before starting electrophoresis, and transferred onto polyvinylidene difluoride (PVDF) membranes. After the membranes were blocked with QuickBlock™ Blocking Buffer for 20 minutes, the membranes were incubated with primary antibody at 4 ℃ overnight, the second day after wash film 3 times, with second antibody for 1 hour. After washing the film again, ECL development is used, and the strip is analyzed. Primary antibody is bax, bcl-2, capase-3 antibody.

1.7 Statistical analysis

A statistical software GraphPad Prism 8.0 was used for statistical analysis. For the analysis of data, logarithmic transformation was performed to normalize the data distribution before conducting ANOVA.Then analyze the data by one-way analysis of variance or Brown-Forsythe and Welch ANOVA tests. The results were statistically analyzed for P<0.05 was considered statistically significant. Data are presented as mean ± SD.

2.1 Morphology

We observed the morphology of hUC-MSCs before and after bilirubin stimulation under microscope (Figure 1A). Normal hUC-MSC morphology are adhered to the wall and grew into relatively uniform spindle cells arranged in parallel. Under the microscope, the number of cells in the visual field decreased with the increase of the concentration of other groups, but the morphological changes were not obvious.

2.2 Cell Viability

First, we studied the effect of bilirubin on cell viability by CCK8 test to determine the time and concentration groups. Results of CCK-8 indicated that with increased concentration of bilirubin would aggravating cell death. With the increased concentration of bilirubin, the viability of hUC-MSCs cultured with bilirubin reduced compared with the control group (Figure 1B). After bilirubin treatment with 0, 5, 10, 15 mg/dL for 12h, the cell viability were (98.56%±1.13%), (36.42 %±2.58%), (21.65%±1.55%), (13.77%±2.06%). And 24h cell viability were (32.93%±1.45%), (21.92%±1.20%), (14.15%±1.38%).

The cell survival rate of each group at 12h is statistically significant. The survival rate of all bilirubin stimulated groups at 12h was significantly lower than that of control group (P<0.001). And there is also statistical significance among the concentration groups (P<0.001). As the concentration increased, the cell survival rate decreased significantly at 12h and 24h. However, there were no significant differences in the the cell survival rate between 12h and 24h in all bilirubin stimulation groups (P>0.05).

2.2 hUC-MSCs Osteogenic Differentiation after bilirubin stimulated

The differentiation ability of hUC-MSCs is the main reason for their initial study and application in treatment. Therefore, we first studied the change of bilirubin intervention on osteogenic differentiation of MSCs (Figure 2A). we can discover that in addition to the 10,15mg/dL group, the cells had undergone morphological changes and overgrown visual fields within 7 days. All groups were covered with 6-well plates at 21 days. Alizarin red S staining (Figure 2B) showed no significant difference in calcium nodules between these groups. This indicates that bilirubin has no effect on osteogenic differentiation.

2.3 hUC-MSCs Adipogenic Differentiation after bilirubin stimulated

hUC-MSCs were differentiated into adipocytes and then were stained with Oil Red O to assess the accumulation of intracellular lipid droplets by electron microscopy. We see that in our experiments, the number of cells was obvious decrease after bilirubin intervention. After 7 days, cells in each group showed obvious morphological changes (Figure 2C), meanwhile cells in high concentration group were significantly less than other groups. At 14 days, the number of cells in all groups increased in addition to morphological changes, but 15mg/dL group cell growth is slow. At 21 days, small lipid droplets could be seen in the cells. After the oil red O staining could be seen in control and 5mg/dL group staining more than 10 mg/dL and 15 mg/dL under electron microscopy (Figure 2D). We can find that the intervention concentration is increased, the cell growth is slow, and the lipop droplets are reduced, however, after removing intervention factors, MSCs still have the function of adipogenic differentiation.

2.4 Cytokines assay after bilirubin stimulated

Il-6 is expressed to varying degrees in a variety of inflammatory responses, as well as in brain injuries treated with hUC-MSCs. In our experiment, cytokine concentration was measured by ELISA. And IL-6 increased significantly after bilirubin stimulation (P<0.05, Figure 3A) had the highest level of IL-6 was found in the 5mg/ml group (18027± 1204 pg/ml) among the three bilirubin stimulation groups (0mg/dL group (6381± 732.6), 10mg/ dL group (15065± 610.1 pg/ml), 15mg/ dL group (14135± 728.7 pg/ml)). And secretion of IL-6 decreased with the increased bilirubin stimulation concentration.

Several demonstrate that hUC-MSCs can modulate the generic pathway of inflammation by introducing negative feedback loops. The hUC-MSCs increase the expression of many factors, including TSG-6, which we are preparing to test. The level of TSG-6 increased after bilirubin stimulation at 12h (0 mg/dL 4.194± 0.3817 ng/ml, 5 mg/dL 6.115± 0.2687 ng/ml, 10 mg/dL 5.623± 0.1035 ng/ml, 15 mg/dL 5.379± 0.1150 ng/ml). TSG-6 had an upward trend after the intervention(P<0.05, Figure 3B). Secretion of TSG-6 also decreased with the increased bilirubin stimulation concentration.

2.4 Apoptosis protein in hUC-MSCs after bilirubin stimulation

We investigated the apoptosis of hUC-MSCs under the influence of bilirubin, and total cell protein was collected. Western blot was used to detect the expression of caspase 3, Bax and Bcl-2 (Figure 4). The increase in the expression of the apoptotic protein Bax, bcl-2 and caspase-3. After treatment with 0, 5, 10, 15 mg/dL bilirubin for 12 h, Bax were respectively (0.7055± 0.1673), (1.020± 0.1348), (1.006± 0.1417), (1.058± 0.1886), caspase-3 were respectively (0.7922± 0.2494), (1.113± 0.1240), (1.071± 0.1230), (1.169± 0.1490), bcl-2 were respectively (0.2719± 0.1146), (0.6827± 0.1368), (0.7402± 0.2028), (0.6546± 0.1196). Compared with 0 mg/dL bilirubin group, the expression of apoptotic protein Bax, bcl-2 and capcase-3 protein were significantly increased in bilirubin group (P>0.05), however there were no significant differences between the intervention groups.

This study sought to investigate the therapeutic potential of MSCs on hyperbilirubinemia in vitro. Hyperbilirubinemia is a common disease in newborns[23]. Too high concentration of bilirubin can lead to acute bilirubin encephalopathy and chronic bilirubin encephalopathy without proper monitoring or treatment, the rapid rising bilirubin in serum can cause neurotoxicity and cause death in patient[1]. The bilirubin neurotoxicity mainly induces apoptosis and necrosis of brain cells, reduces oxygen consumption of neurons, and increases the release of calcium, inflammatory factors and apoptotic proteins[24]. In addition, UB has different degrees of adverse reactions to plasma, mitochondria and endoplasmic reticulum [25, 26]. These changes will eventually lead to excitotoxicity induces neuronal cell swelling dueto excessive influx of Na⁺, Ca²⁺, Cl⁻ and water, triggering cell death by both apoptosis and necrosis causing acidosis and aggravating brain damage[23, 27]. Some scholars have found that unbound bilirubin can also act as an immune stimulus factor, inducing the activation of microglia in vivo, up-regulating the expression of pro-inflammatory genes, triggering the release of inflammatory factors and producing a pro-inflammatory environment. The production of pro-inflammatory cytokines can aggravate bilirubinin-induced neuronal apoptosis and necrosis [28, 29]. Phototherapy and blood exchange are the main treatment of severe hyperbilirubinemia. Due to the rapid development and severity of bilirubin encephalopathy, there are no good treatments for neurological sequelae[4, 30]. The current treatment need to be updated and improved, and new treatment methods are also being explored.

Now stem cell therapy is attracting the attention of many scholars. As a cell therapy, MSCs therapy is different from drug therapy. It mainly relies on the differentiation potential of cells and secretes a variety of cytokines and active molecules to play a therapeutic role. Hence, the survival of MSCs was crucial to exert better therapeutic efficacy in diseases. It is imperative to observe the survival status of MSCs in bilirubin intervention. In this study, we found apoptosis of MSCs increased after bilirubin stimulation. And we could find that the number of survival cells goes down with the bilirubin concentration going up. The survival rate of MSCs also decreased (Difference was statistically significant, p < 0.05). This suggests that if we use MSCs to treat patients with different levels of bilirubin, the same dose can have a different effect. The expressions of apoptosis proteins Bax, Bcl-2 and CAPCASE-3 in bilirubin group were significantly increased (P > 0.05), suggesting that bilirubin may induce apoptosis of MSCS through bcl-2 apoptosis protein and Caspase3 signaling pathway. It has been reported that treatment of developing rat brain neurons with purified unbound bilirubin results in reduced mitochondrial transmembrane potential, resulting in the release of cytochrome C, and the transfer of Bax (an apoptosis promoter) to the mitochondrial membrane may inactivate the apoptosis inhibitor Bcl-2 and form selective ion pores on the membrane. This is another mechanism that allows cytochrome C to escape. The cytosolute cytochrome C then binds to apoptotic protease activator − 1 (APAF-1), triggering activation of Caspase-9. Caspase-9 cleaves and activates caspase-3 into its active form to perform the cell death process (apoptosis cascade) [4]. Therefore, in clinical use of stem cell therapy, neonatal bilirubin level and the possible toxic effect on stem cells should be fully considered, and more doses of stem cells may be needed for hyperbilirubinemia in children to achieve the therapeutic effect.

MSCs can be isolated from autologous and expanded in vitro. As has been widely acknowledged, MSCs can differentiate into several directions, including osteogenic, adipogenic, chondrogenic and neurogenic differentiation[31, 32]. We investigated the adipogenic and osteogenic differentiation of MSCs stimulated by bilirubin. It was found that bilirubin reduced the proliferation ability of MSCs but had little effect on differentiation function. This proves that serum bilirubin concentration does not affect its differentiation when they are used for treatment. Researches showed that MSCs also exhibited immunosuppressive or immunomodulatory properties. Part of its immunity is accomplished through the paracrine pathway[33]. This property makes MSCs treatment a promising approach for repair of neonatal brain injury. Basic researches have illustrated that MSCs could alleviate neonatal brain injury in animal models[10, 13, 16, 21]. At present, there have been a number of studies for MSCs to treat brain injury, but there are few studies on the use of MSCs to treat bilirubin encephalopathy. However, there are still many problems to be studied before MSCs can be used in clinical treatment. And First of all, we know the bilirubin have toxic effect on cells, between the bilirubin on MSCs may also produce toxic effect. Our experiment mainly discussed whether the MSCs used for treatment are affected by the unconjugated bilirubin in patients and whether they still have therapeutic potential. Our findings offer high concentration bilirubin has toxic effect on MSCs and damage its function in vitro. To investigate the role of MSCs after bilirubin stimulation is conducive to exploring whether MSCs therapy is combined with other treatment methods or only treated after reducing serum bilirubin concentration. It can suggest possible therapeutic method for clinical of neonatal bilirubin encephalopathy.

In recent years, studies have found that MSCs can also play immunosuppressive or immunomodulatory roles in the treatment, and their immunity is accomplished through the paracrine pathway. There is increasing evidence that MSCs rely on the secretion of various cytokines or active molecules to play a protective role in cerebral diseases[34]. It has been reported that IL-6 secreted by MSC can reduce inflammation. In the mouse model of myocardial infarction, MSC activates M2 macrophages through secretion of IL-6[35–37]. Our data showed that IL-6 secreted by hUC-MSCs increased after bilirubin stimulation, but as the concentration of bilirubin increased, the IL-6 decreased, it indicating that although bilirubin has an immunostimulatory effect on hUC-MSCs, it also impairs the paracrine function of hUC-MSCs to a certain extent. Moreover, TSG- 6 is an inflammation-inducing protein that can reduce tissue inflammation and promote damaged tissue repair in inflammatory diseases. Meanwhile, some studies have shown that TSG-6 also could promotes M2 macrophage polarization[11, 38]. And in our experiment TSG-6 had an increasing trend in each group after stimulation. However, TSG-6 also decreases after the concentration increases. We found that the secretion of TSG-6 was increased after bilirubin stimulation, but the secretion of TSG-6 was decreased with increased bilirubin concentration. Therefore, we speculated that the secretion function of MSCs would decrease with the injury of MSCs. The secretion of different cytokines can be affected under different conditions. Meanwhile, we know that IL-6, as a soluble substance secreted by MSCs, can further activate signal transduction and activating factors, which in turn causes the down-regulation of the pro-apoptotic protein Bax protein and exerts an anti-apoptotic effect[39]. These results suggest that moderate levels of bilirubin can stimulate MSCs to secrete cytokines, play anti-inflammatory, anti-apoptotic and promote repair. High concentrations of bilirubin inhibit paracrine function. Therefore, under the stimulation of high bilirubin concentration, the number of stem cells decreases, the differentiation function is impaired, and the paracrine function will further decline, which is not conducive to the repair of tissue cells.

Taken together, we speculated that bilirubin may have toxic effects on umbilical cord mesenchymal stem cells, and with the increase of bilirubin level, the proliferation and paracrine function of umbilical cord mesenchymal stem cells will be further affected. But it had no effect on stem cell differentiation. These results suggest that when considering the use of stem cells for the treatment of neonatal nervous system injury, the possible effect of neonatal serum bilirubin level on stem cells should be considered. On the one hand, a larger dose of stem cells may be needed. On the other hand, it is necessary to take therapeutic measures to rapidly reduce bilirubin, so as to reduce the toxic effect of bilirubin on stem cells and affect their therapeutic effect. This study provides experimental basis for the treatment of bilirubin encephalopathy with mesenchymal stem cells. The specific methods and dosage of mesenchymal stem cells in clinical application remain to be further studied.

Acknowledgements

We thank Chongqing Engineering Research Center of Stem Cell Therapy.

Authors’ contributions

Zhang Yanqi ,Liu Jiang, Wei Hong and Ai Qing wrote the main manuscript text and Hu Ya prepared figures 1-2,Sun Qian andJi Yanchun prepared figures 3-4. All authors reviewed the manuscript.

Funding

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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