1. Purification and culture of P1 MSC
The MSCs were isolated from adipocytes as described in materials and methods. The morphology of MSCs are shuttle-shaped as shown in the Figure 1. To confirm and identify the MSCs, cells isolated from adipocytes were stained with anti-CD90 and CD45 antibodies after 48 hours culturing, and detected by flow cytometry. CD90 was used as positive surface marker for MSC and CD45 was used as a negative marker. CD90 was highly expressed in our isolated cells, while CD45 was merely presented. We further investigated the expression levels of CD90 marker from passage 1 to passage 4, and found that passage 1 cells expressed the highest level of CD90 than other generations (94.9% positive in P1, and was followed by P2-93%, P3-79.4%, P4-69.9 %). Therefore, we used the cells from passage 1 for the rest of experiments.
2. L-theanine reduces free radical & promote viability
There were various reports indicated that tea extracts have biological and pharmacological activities such as antioxidation 32, anti-carcinogenesis
33, 34, anti-mutagenesis 35, and anti-inflammation 36. L-theanine is a free amino acid in tea extracts. It was reported to act as a neurotransmitter in the brain and has a relaxation-inducing effect in humans 37-40. According to previous evidences, we presumed that the ingredients in tea extracts such as EGCg and L-theanine may strengthen the therapeutic effects of MSCs on damaged tissue. In order to improve the therapeutic effects of MSCs, we first pretreated the MSCs with EGCg and L-theanine. The level of free
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radicals of fresh medium, MSC-conditioned medium followed by measuring the free radical levels of EGCg-pretreated MSC-conditioned and L-theanine-pretreated MSC- conditioned medium by Chemiluminescence Analyzing System. We observed the significant reduction of free radical levels in all of the MSC-conditioned medium (Figure 2A). Moreover, the viability of MSC cells was improved by EGCg or L-theanine treatments regardless the treatment concentrations (Figure 2B). We used 10 µM as a moderate concentration of L-theanine pretreatment in the following experiments.
3. L-theanine preconditioning promotes MSC migration
To evaluate the effects of L-theanine on the migration of MSCs, the wound healing assay were performed and the migration situation of MSCs in different concentration of L-theanine precondition were analyzed. It is obviously that L-theanine preconditioned MSCs migrated faster than the control group (Figure 3).
4. L-theanine promotes liver regeneration and angiogenesis
The MSC-conditioned medium and L-theanine preconditioned medium were collected and analyzed for the secretion of cytokines. The expression of IFNg and VEGF were shown in Figure 4. IFNg, angiotensinogen, and caspase 12 are markers which highly compatible to liver regeneration 41, 42. In previous study of suppression on hepatic stellate cell activity, antioxidant such as alpha-tocopherol (vitamin E), γ-interferon, and hepatocyte growth factor (HGF) all had the inhibit ability 43 .We observed that 10T group (10 μM L-theanine preconditioned MSCs) showed a higher expression of IFNg then the MSCs only control group
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(MSC-conditioned medium) and medium only control. VEGF plays a role on angiogenesis, and its expression revealed about 20 times higher in 10T group than the control group (Figure 4).
5. Liver function is significantly recovered after MSC treatment
To investigate the therapeutic effects of MSC in vivo, we generated a DEN-induced liver-injury rat model. The experimental flow chart was presented in Figure 5. The liver tissues were collected and the appearances were observed (Figure 6). The serum activity of liver function indicators (AST, ALT, and γ-GT) were determined by a chemistry analyzer. Liver markers were significantly increased in DEN-groups as the time prolonged after DEN treatment, and MSC treatments significantly decreased these markers, except for γ-GT (Figure 7).
6. MSC treatment reduces ROS amount
To further evaluate the MSC effects on ROS production, we measured the ROS levels in the rat whole blood. Results indicated that the levels of ROS augmented as the time progression after DEN treatment. After MSC treatments, the levels of ROS were decreased compared to the DEN only, not in MSC treatment groups (Figure 7D).
7. H-E staining shows the liver injury status of each group
To reveal the pathologic evidences of MSC therapeutic effects, all liver tissues were collected and subjected to hematoxylin-and-eosin (HE) staining (Figure 8A). The increased shattered tissue was noted in the DEN-induced groups but not in sham control groups. Oppositely, the shattered situation decreased in MSC treatment groups. H-E staining of portal triad was also displayed in Figure 8B. A portal triad, as known as portal field,
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portal area, or portal tract, is a distinctive arrangement in liver, consisting proper hepatic artery, hepatic portal vein, and small bile ductules of cuboidal epithelium.
8. MSC treatment reduces fibrosis, apoptosis, but not pyroptosis and autophagy in DEN-treated livers
To explore whether DEN affected hepatic fibrosis, we investigated the fibrosis status using Masson staining for connective tissues (Figure 8C).
To confirm the DEN-induced injury stages, we further stained the tumor marker, alpha-fetoprotein (AFP) (Figure 8D). To investigate how did DEN influence liver injury, the apoptosis, autophagy, and pyroptosis markers were also detected, including the following biomarkers, Bax, Bcl-2, caspase 3, Beclin-1, LC-3β, caspase 1, and IL-1β. The results showed a significant inhibition of apoptosis in MSC treatment groups and L-theanine preconditioned groups after DEN-induced liver injury. However, in autophagy and pyroptosis, there were no significant changes observed in both MSC treatment groups and L-theanine preconditioned groups.
9. Western blot analysis conforms that MSC treatment inhibits apoptosis, but not pyroptosis and autophagy in DEN-treated livers
To confirm the pathology of IHC (immunohistochemistry) results, hepatic levels of apoptosis-related proteins (Figures 9, 10 and 11), pyroptosis-related proteins (Figures 12 and 13), and autophagy-related proteins (Figures 14 and 15) were determined by Western blot analysis.
The results showed marked increases in the expression of Bax and caspase 3 in the DEN-induced groups, while the expression levels of Bcl-2 were decreased. These observations were absent in the MSC treatment groups,
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which indicate that MSCs have the function to inhibit apoptotic formation.
There were no significant differences for caspase-1 and IL-1β expressions in each group. The expression of autophagy-related proteins Beclin-1 and LC-3β was significantly elevated in DEN-induced groups. However, there were no significant effects on Beclin-1 and LC-3β expression in MSC treatment groups and L-theanine preconditioned MSC groups.
10. MSCs and tumor in vivo were traced
Caliper IVIS system is a proper method to trace the MSCs delivery and their interactions with tumors in vivo. We used 2-DG as a tumor marker to detect the existing of tumors in DEN-induced rats (Figure 16A).
Indicated in Figure 16B, control rats and DEN-induced rats were treated with DiR fluorescence-labeled MSCs 2 hours before photographed by IVIS.
DiR is a lipophilic, near-infrared fluorescent cyanine dye. Results showed that the majority distribution of 2-DG in 8-week of DEN-treated rats were in liver under IVIS detection (Figure 16A). The distribution of MSCs 2 hours after intravenous transplantation showed majority in the bladder but some focused in liver compared to no DEN-damaged group (Figure 16B).
These results indicate that DEN-induced liver dysfunction triggers liver to release messages and elevates MSCs homing effects to impaired tissues.
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