Figure 1. Characterization of adipose tissue-isolated MSCs.
Primary rat MSCs isolated from adipose tissue were stained with (a) a conjugated anti-rat CD90 monoclonal antibody or (b) a FITC-conjugated anti-rat CD45 monoclonal antibody. The percentage of CD90 positive cells in different passages of (c) primary MSC cultures. (d) In passage 1 of rat MSCs, to the percentage of CD90 positive cells were the highest one among the first four passages of primary cultured MSCs.
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(a)
(b)
Figure 2. ROS counts decrease in MSC conditioned medium pretreated with EGCg or L-theanine.
(a) ROS counts in MSC-conditioned medium, EGCg-preconditioned medium and L-theanine-preconditioned medium were all significantly decreased compared to medium only. (b) Cell viability in different concentrations of L-theanine of MSC-conditioned medium was represented as the mean ± SEM. *p<0.05 versus control group. One-way-ANOVA followed by student’s t-test. ROS counts were significantly decreased in MSC-conditioned medium.
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Figure 3. L-theanine promotes MSC migration.
(a) MSCs were pretreated with EGCg or L-theanine and the wounds were created with a 10 μL tip. The migration situations of MSCs were evaluated after 24 hours of culturing. L-theanine preconditioned-MSCs migrated faster than other two groups. (b) Quantitative analysis of the level of migrated MSCs. Values are presented as mean ± SD. *p<0.05 compared with control group.
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(a) (b)
Figure 4. L-theanine increases immunomodulatory function and angiogenesis.
(a) Secreted levels of IFNg or (b) VEGF in the medium only or MSC-conditioned medium with or without EGCg or L-theanine were detected by ELISA. L-theanine pretreatment increased the secretion of IFN- and VEGF.
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Figure 5. DEN-induced liver injury in the rat model. The experimental flow chart of the rat model establishment. Liver injury was induced by DEN in the 7-week-old rats. 1×106 cells/rat MSCs or L-theanine preconditioned MSCs were given to rat by intravenous injection after 2, 4 or 8 weeks of DEN injury. Liver tissue and blood were collected at the indicated time points for further evaluation.
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Figure 6. Morphology of liver tissues from 10 groups. Appearance of liver tissues from Wistar rats with DEN and MSC treatments. The rats were sacrificed and the liver were collected for photographing. Festered tissues were seen on 8 weeks of DEN-induced liver.
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(a) (b)
(c)
Figure 7(a)(b)(c) Biochemical markers of liver and bile ductules function.
(a)(b) DEN treatment increased the AST, ALT activity in serum (*p<0.05), while MSC or L-theanine preconditioned-MSC treatment decreased AST and ALT activity. (c) γ-GT value was significant enhanced in DEN-induced groups, there was a decreasing trend in MSC treatment groups but no significant differences were shown.
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(d)
Figure 7(d). MSCs decrease levels of ROS on DEN-induced rats.
(d) Whole blood was collected after sacrificed and then the level of ROS was detected by Chemiluminescence Analyzing System. It was apparently notified that MSC treatment groups and L-theanine preconditioned-MSC groups reduced the levels of free radical induced by DEN treatment. (Mean
± SEM; n=6/group). In conclusion, we observed severe liver injury after DEN treatment but relieved situation after MSC application.
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(a)
Figure 8(a). Histological evaluation by H-E staining (400x)
All livers were collected after sacrificed under anesthesia, fixed with formalin and waxed. We de-waxed and hydrated livers in paraffin sections, and stained the slides in hematoxylin for 5 minutes and immersed in eosin for 30 seconds, then dehydrated by xylene.
(a) Livers of the rats administered only DEN showed shattered images,
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while livers in MSC treatment groups showed more normal appearance of liver parenchyma.
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(b)
Figure 8(b). H-E staining of liver portal triad (400x)
(b) Image shows occlusion of bile ductule in DEN-induced groups. (Red arrow: hepatic artery, Blue arrow: hepatic portal vein, Black arrowhead:
small bile ductule) Hepatic sections were re-fix in Bouin's solution, and stain in Weigert's iron hematoxylin working solution, then stain in Biebrich scarlet-acid fuchsin solution. Phosphomolybdic-phosphotungstic acid
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solution, aniline blue solution, acetic acid solution were orderly applied then clear in xylene.
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(c)
Figure 8(c). Masson staining of liver tissues (400x)
(c) In fibrosis-specific Masson staining, the degree of hepatic fibrosis in DEN-induced group was observed.
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(d)
Figure 8(d). High expressions of AFP were observed in DEN-induced groups (400x)
(d) In general, HCC tissue or serum level frequently expresses high level of α-fetoprotein (AFP). We detected higher expressions of AFP in DEN treatment groups (red arrow), which indicated that we successfully established the DEN-induced carcinoma model.
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Figure 9. Relationships between apoptosis-related protein Bax and DEN or MSC treatment. Liver tissue slices obtained as described in figure 9 were subjected to (a) IHC staining for detecting Bax (Red arrow, 400x). The positive staining signals were measured by ImageJ and the quantitative results were showed in (b). (Mean ± SEM; n=6/group;
*p<0.05 versus control group; a<0.05 versus 2 week of DEN-induced group; b<0.05 versus 4 week of DEN-induced group; c<0.05 versus 8 week of DEN-induced group) Liver tissue extracts were also analyzed by Western blot for the protein levels of (c) Bax. The expression of Bax were increased significantly after DEN treatment and decreased with the MSC treatment. Data was showed in mean ± standard deviation (SD) of 3-5 separate experiments.
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Figure 10. Relationships between apoptosis-related protein Bcl-2 and DEN or MSC treatment. Liver tissue slices obtained as described in figure 10 were subjected to (a) IHC staining for detecting Bcl-2 (Red arrow, 400x). The positive staining signals were measured by ImageJ and the quantitative results were showed in (b). (Mean ± SEM; n=6/group;
*p<0.05 versus control group; a<0.05 versus 2 week of DEN-induced group; b<0.05 versus 4 week of DEN-induced group; c<0.05 versus 8 week of DEN-induced group) Liver tissue extracts were also analyzed by Western blot for the protein levels of (c) Bcl-2. The expression of Bcl-2 were decreased significantly after DEN treatment and increased with the MSC treatment. Expression of Bcl-2 (red arrow) analyzed in IHC or Western blot all showed opposite pattern to Bax. Data was showed in mean
± standard deviation (SD) of 3-5 separate experiments.
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Figure 11. Relationships between apoptosis-related protein caspase 3 and DEN or MSC treatment. Liver tissue slices obtained as described in figure 11 were subjected to (a) IHC staining for detecting caspase 3 (Red arrow, 400x). In caspase 3 IHC results, it was similar to the Bax expression, MSC application significantly reduced the expression of caspase 3 (Figure 9c). This indicated that DEN treatment induced apoptosis and MSC treatments inhibited the apoptotic process. The positive staining signals were measured by ImageJ and the quantitative results were showed in (b).
(Mean ± SEM; n=6/group; *p<0.05 versus control group; a<0.05 versus 2 week of DEN-induced group; b<0.05 versus 4 week of DEN-induced group; c<0.05 versus 8 week of DEN-induced group) Liver tissue extracts were also analyzed by Western blot for the protein levels of (c) caspase 3.
The expression of caspase 3 were increased significantly after DEN
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treatment and decreased with the MSC treatment. Data was showed in mean ± standard deviation (SD) of 3-5 separate experiments.
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Figure 12. Relationships between pyroptosis-related protein caspase 1 and DEN or MSC treatment. Liver tissues slices were subjected to IHC staining for pyroptosis markers (a) caspase 1 (Red arrow, 400x). The quantitative results were showed in (b). Western blot analysis of liver tissue extracts was also applied to detect the expression levels of (c) caspase 1.
Western blot analysis reveals DEN induced liver injury by pyroptosis, whereas the MSC treatment and L-theanine preconditioned MSC seems to reduce the pyroptosis-related protein expression. (Mean ± SEM; n=6/group;
*p<0.05 versus control group; c<0.05 versus 8 week of DEN-induced group)
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Figure 13. Relationships between pyroptosis-related protein IL-1β and DEN or MSC treatment. Liver tissues slices were subjected to IHC staining for pyroptosis marker (a) IL-1β (Red arrow, 400x). The quantitative results were showed in (b). Western blot analysis of liver tissue extracts was applied to detect the expression levels of (c) IL-1β. Western blot analysis revealed DEN also induced liver injury by the pyroptosis pathway. MSC treatment seems to reduce the pyroptosis-related proteins expression. (Mean ± SEM; n=6/group; *p<0.05 versus control group;
c<0.05 versus 8 week of DEN-induced group)
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Figure 14. Relationships between autophagy-related protein Beclin-1 and DEN or MSC treatment. Liver tissues slices were subjected to IHC staining for autophagy marker (a) Beclin-1 (Red arrow, 400x). The quantitative results were showed in (b). In Beclin-1 IHC results, MSC application significantly reduced the expression of Beclin-1. This indicated that DEN treatment induced autophagy and MSC treatments inhibited the autophagy process. Western blot analysis of liver tissue extracts was also applied to detect the expression levels of (c) Beclin-1. In western blot analysis, there was a trend in increasing expression in DEN-induced groups and reducing in 4 weeks of DEN treatment and MSC and in L-theanine preconditioned groups. (Mean ± SEM; n=6/group, *p<0.05 versus control group; c<0.05 versus 8 week of DEN-induced group)
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Figure 15. Relationships between autophagy-related protein LC-3β and DEN or MSC treatment. Liver tissues slices were subjected to IHC staining for autophagy marker (a) LC-3β (Red arrow, 400x). The quantitative results were showed in (b). Western blot analysis of liver tissue extracts was also applied to detect the expression levels of (c) LC-3β. The expression of LC-3β was increased significantly after DEN treatment and decreased with the MSC treatment. (Mean ± SEM; n=6/group; *p<0.05 versus control group; a<0.05 versus 2 week of DEN-induced group; b<0.05 versus 4 week of induced group; c<0.05 versus 8 week of DEN-induced group)
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(a)
Figure 16(a). IVIS system tracks and images tumor in vivo.
(a) IVIS imaging of rats in control group and 8 weeks of DEN treatment after tumor marker AFP injection into the tail vein. The liver of 8-week DEN treated rat was directly imaged and was characterized with a high expression of AFP. Tumor site was indicated by the blue fluorescence.
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(b)
Figure 16(b). IVIS system tracks and images MSC in vivo.
(b) MSCs were labeled with DiR surface marker and were injected into the tail vein. MSC homing ability revealed the damaged liver site indicated by the blue fluorescence.