Effect of Youthful Blood Environment and Its Key Factor SCF on Renal Interstitial Fibrosis in Elderly Mice

Background: youthful blood environment was shown to decelerate the aging process of kidney and to attenuate senile renal �brosis in a young-old parabiotic animal model; in addition, we identi�ed a stem cell factor (SCF) that is closely linked with the process. To further investigate the effect of youthful blood environment on renal interstitial �brosis and the underlying mechanisms, we bred SCF receptor c-Kit gene loss-of-function Wps/Wps mice and established a combination mice model that was subjected to unilateral ureteral obstructive (UUO) and parabiotic surgeries. Methods: Parabiotic mice were divided into isochronic parabiotic (young-young, Y-IP and old-old, O-IP) and heterochronic parabiotic (young-old, HP) groups. UUO surgery was performed in one of the parabiotic pairs in the IP group (Y-IPuuo and O-IPuuo) and in the elderly mice in the HP group (O-HPuuo). In order to study the role of SCF/c-kit on renal interstitial �brosis, UUO surgery was performed in wildtype (WT) and Wps/Wps mice. Results: Fourteen days after UUO surgery, the kidney interstitial �brosis area, kidney function, and the expressions of SCF/c-Kit, pNF-κB, and brosis-related proteins in the O-HPuuo group were signi�cantly lower than those in the Ouuo and O-IPuuo groups. Compared with wildtype UUO mice, the expressions of pNF-κB and brosis-related proteins, kidney interstitial �brosis area, and the kidney function were all signi�cantly decreased in Wps/Wps UUO mice. Conclusions: Youthful blood environment downregulated the expressions of SCF/c-Kit in elderly UUO mice, and ameliorated UUO-induced kidney �brosis and function loss, which may be mediated via the NF-κB pathway.


Background
Renal interstitial brosis is the basis of renal senescence, and is one of the most common mechanisms of deterioration of kidney disease into end-stage renal disease.Therefore, effective control or reversal of renal brosis is the key to prevent renal senescence and deterioration of a variety of kidney diseases [1][2][3].
Parabiotic animal model is characterized by shared blood circulation established via surgical linkage between the muscle ap and skin of two animals.Parabiotic animal models are categorized into isochronic parabiotic (IP) and heterochronic parabiotic (HP) models, depending on the age of the two parabiotic animals.Parabiotic animal models have been gradually applied in recent years for the study of senescence of heart [4], liver [5], nerves [6,7], and muscle [8] tissues.The results have consistently indicated that youthful blood environment can promote proliferation, reduce tissue injury, and help recover organ function to varying degrees.
In our previous study, we observed lower expressions of renal senescence markers and decreased kidney brosis area in elderly HP mice after 5 weeks of shared blood circulation, but with no obvious change in renal function [9].Furthermore, the expressions of renal senescence markers were decreased 16 weeks after transplantation of elderly rat kidney to young rat; however, no obvious changes were observed with respect to kidney brosis and renal function [10].Kidney tissue damage and function loss were signi cantly alleviated in elderly HP mice after ischemia-reperfusion injury (IRI), compared to the control old IRI mice [11].However, the effect of youthful blood environment on renal interstitial brosis and the associated underlying mechanisms are not clear.In addition, in earlier studies, we compared serum cytokine levels in young and elderly mice with 5 weeks of IP or HP, and found that 6 types of blood factors including stem cell factor (SCF) may be associated with renal senescence; in addition, we also con rmed higher expressions of SCF in elderly mice blood.
Stem cell factor is also referred to as the mast cell growth factor (MGF) or c-Kit ligand (KL).It has been shown to promote cell proliferation, mobilization, and adhesion [12].Early studies about SCF largely pertained to the treatment of blood system diseases [13].Several recent studies have indicated that SCF/c-kit plays a critical role in the process of reconstruction and brosis in a variety of diseased organs, such as lung, skin, liver and kidney (14)(15)(16)(17).SCF is associated with development of renal brosis in multiple kidney diseases.Moreover, SCF is highly expressed in a variety of kidney diseases, such as IgA nephropathy, membranous nephropathy, crescent nephritis, hypertensive nephropathy, diabetic nephropathy, late AKI, and graft-versus-host reaction after kidney transplantation (18-20).However, the role of SCF in elderly renal brosis and the associated mechanisms are not clear.
In this study, by establishing a combination mice model with unilateral ureteral obstructive (UUO) and parabiotic surgeries, we investigated the effect of youthful parabiotic circulation on senile renal interstitial brosis and studied the associated mechanisms.In order to understand the role of SCF/c-Kit on renal interstitial brosis, the UUO model was also established using Wps/Wps mice with loss-of-function mutation in the SCF receptor c-Kit.

Establishment of animal model
Young (age: 3 months) and elderly (age: 22 months) SPF graded mice were obtained from the Experimental Animal Center at the Chinese PLA General Hospital.Three-month-old Wps/Wps mice with a C57BL/6 strain background were obtained from the MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University.The methods for parabiosis surgical procedures and the veri cation of shared circulation in parabiotic mice are described elsewhere [9].For establishment of unilateral ureteral obstruction (UUO) mice model, the left ureter was ligated using 5 − 0 silk via a midabdominal incision.All surgical procedures were performed under iso urane anesthesia.Mice were sacri ced 7 or 14 days after UUO surgery.The contralateral kidneys were harvested as internal controls.
All animal experiments were approved and performed in accordance with the Chinese PLA General Hospital's Committee on Animal Protection and Utilization.

Experimental animal groups
In order to study the effect of blood environment on senile kidney interstitial brosis, experimental animals were divided into parabiotic and normal mice groups.Normal mice group comprised of young control group (Ycon) and old control group (Ocon).Parabiotic mice group comprised of isochronic parabiotic (IP) and heterochronic parabiotic (HP) groups.Young-young (Y-IP) and old-old (O-IP) pairings were performed in the IP group, while young-old pairing was performed in the HP group.Three weeks after parabiosis, UUO surgery was performed in one of the parabiotic pairs in the IP group and in the elderly mice in the HP group.According to duration of UUO obstruction, these mice were divided into 7 d and 14 d groups: young control groups (Yuuo7 and Yuuo14); old control groups (Ouuo7 and Ouuo14); Y-IP groups with UUO in the young mice (Y-IPuuo7 and Y-IPuuo14); O-IP groups with UUO in the old mice (O-IPuuo7 and O-IPuuo14); HP groups with UUO in the old mice (O-HPuuo7 and O-HPuuo14).
In order to study the role of SCF/c-kit on renal interstitial brosis, UUO surgery was performed in 3-month old wildtype (WT) and Wps/Wps mice.These mice were divided into WTuuo7, WTuuo14, Wps/Wpsuuo7, and Wps/Wpsuuo14 groups, according to the length of the postoperative period.

Blood biochemistry analysis
Mouse serum creatinine and blood urea nitrogen (BUN) levels were determined using a Hitachi 7150 automatic biochemistry analyzer.Mouse serum SCF was examined using Quantikine ELISA Kit (R&D Systems) according to the protocol.

Histopathological examination
Kidney tissues were xed using 10% neutral-buffered formalin.The kidney tissue blocks were embedded with para n and sections stained with periodic acid-Schiff (PAS).The degree of brosis in young and old kidney samples were determined using Sirius-red staining and Masson's Trichrome staining, as described elsewhere [21].

Immuno uorescence
Frozen young and old kidney sections were rst blocked with 5% bovine serum albumin (BSA) in PBS for about half an hour, and incubated overnight with primary antibodies anti-α-SMA and anti-TGF-β1 at 4 °C.After washing with PBS, the sections were incubated with secondary antibodies goat anti-rabbit IgG (H + L) antibody (Alexa Fluor-488, Invitrogen) and goat anti-Mouse IgG (H + L) antibody (Rhodamine RedTM-X, Jackson ImmunoResearch) for 2 h in dark at room temperature.Cell nuclei were stained with 1 µg/mL Hoechst for about 5 min.Confocal images of mouse kidney section were obtained using FluoView FV10i Confocal Laser Scanning Microscope (Olympus, Tokyo).

Statistical analysis
All data analyses were performed using SPSS v21.0 software.Normally distributed quantitative data were presented as mean ± standard deviation (SD).Enumeration data are expressed as percentage.For assessing statistical signi cance, the changes of quantitative data among groups were tested by oneway ANOVA; inter-group differences were analyzed using post hoc Dunnett-t test.The Chi-squared test was used to analyze the changes in enumeration data.Values of P < 0.05 were considered statistically signi cant.

Survival condition of parabiotic mice after UUO surgery
No perioperative deaths occurred after UUO surgery of parabiotic mice with shared circulation for 3 weeks.One young-old paired and 1 old-old paired mice without UUO surgery died 1 week later, which suggested that the animal deaths were likely related with the parabiotic surgery but not with the UUO treatment.Two weeks after UUO surgery, death of 1 pair each occurred in the young-young paired, old-old paired, and young-old paired groups.Of these, the receptor mouse died in the young-young paired group, both donor and receptor mice died in old-old paired and young-old paired groups.The survival rate was high in UUO surgery groups, and the death rate in the young-young paired groups was slightly lower than that in the other groups (Table S1).Two weeks after UUO surgery, the survival rate was 100% for the control young or old mice groups.Finally, there were 4 pairings of Y-IPuuo7, O-IPuuo7 and O-HPuuo7 mice, respectively; 5 pairings of Y-IPuuo14 mice; 4 pairings of O-IPuuo14 and O-HPuuo14 mice, respectively.

Youthful blood environment promotes renal function recovery in old mice after UUO surgery
The serum creatinine levels of young UUO mice showed a signi cant increase to 1.92 times of the baseline at 7 days after surgery, remained unchanged from 7-10 days after surgery, and subsequently showed a signi cant decrease to 1.49 times of the baseline at 14 days after surgery.A similar trend was observed in the BUN levels; the BUN level reached a peak at 7 days after surgery (2.18 times of baseline level) and then decreased to 1.72 times of the baseline level at 14 days after surgery.The serum creatinine levels of old UUO mice showed a signi cant increase to 1.43 times of the baseline at 7 days after surgery, and continued to rise to 1.87 times of baseline at 14 days after surgery.The BUN level also maintained the increasing trend, and increased to 2.37 times of the baseline at 14 days after surgery (Figure S1C).There was no signi cant difference with respect to serum creatinine or BUN peak levels between the old and young mice at 14 days after UUO surgery.The only difference was that no sign of slowing down was observed in the old UUO mice groups (Figure S1A, S1B).
The trend of serum creatinine and BUN levels in Y-IPuuo and O-IPuuo groups was identical or a slightly lower than that in the Yuuo and Ouuo groups, respectively.The serum creatinine levels in the O-HPuuo7 and Ouuo7 groups increased to 1.44 and 1.43 times of baseline value, respectively.However, 14 days after UUO surgery, the serum creatinine level in the O-HP mice remained at a level (1.49 times baseline value) similar to that at 7 days after surgery, which was signi cantly lower than that in the Ouuo14 (1.87 times baseline value) and O-IPuuo14 (1.79 times baseline value) groups.The trend of BUN level in O-HP mice was similar to that of serum creatinine level.The BUN level in the O-HPuuo14 group (1.83 times baseline value) was signi cantly lower than that in the Ouuo14 and O-IPuuo14 groups (2.23 times and 2.45 times baseline values, respectively) (Figure S1D-F).

Youthful blood environment alleviates renal tissue injury of elderly UUO mice
Histological examination of kidney tissues showed renal tubular epithelial cell necrosis and shedding, in ammatory cell in ltration and brosis in the Yuuo7 group.The degree of pathological damage in the Ouuo7 and Yuuo7 groups was similar.However, 14 days after surgery, Ouuo14 group showed more obvious renal tubular epithelial cell necrosis, formation of casts, and interstitial brosis than that in the Yuuo14 group (Figure S2).
The pathological manifestations in parabiotic UUO mice were similar to those in the control UUO mice of the same age, i.e., there was no signi cant difference between Y-IPuuo and Yuuo groups or between O-IPuuo and Ouuo groups (images not shown).The extent of pathological damage in the O-HPuuo7 group was similar to that in the O-IPuuo7 and Ouuo7 groups (Figure S2).The trend of kidney damage in O-HPuuo14 was comparable to that in Y-IPuuo14 or Yuuo14 group, i.e., the O-HPuuo14 group did not exhibit aggravation of renal tubular epithelial cell necrosis and shedding.In addition, the extent of interstitial brosis was signi cantly lower than that in the O-IPuuo14 and Ouuo14 groups (Figure S2).These ndings indicate that youthful blood environment during parabiosis alleviated kidney tissue damage in elderly UUO mice.

Youthful blood environment alleviates renal interstitial brosis in elderly UUO mice
The expressions of brosis-associated proteins such as ColI, α-SMA, and TGF-β1 in the Ouuo14 group were signi cantly higher than those in the Yuuo14 group.These protein expressions in the Y-IPuuo14 and O-IPuuo14 groups were similar to those in the Yuuo14 and Ouuo14 groups, respectively.The expressions of these three proteins in the O-HPuuo14 group were signi cantly lower than those in the O-IPuuo14 and Ouuo14 groups, and similar to those in the Y-IPuuo14 and Yuuo14 groups (Figure S2).These results indicate that youthful blood environment may alleviate renal interstitial brosis in elderly UUO mice.

Blood factor SCF expression is closely related to increasing age and renal interstitial brosis
The serum SCF expression in elderly mice was signi cantly higher than that in young mice, as indicated by ELISA assay (Fig. 1A).The serum expression of SCF in healthy people exhibited a positive correlation with age (Fig. 1B).The expressions of SCF and its receptor c-Kit in elderly mice kidney tissues were signi cantly higher than those in young mice (Fig. 1C, 1D).
In order to verify the role of SCF/c-Kit in renal interstitial brosis, the expressions of SCF and its receptor c-Kit were determined at 7 and 14 days after UUO surgery.Compared with the sham group, the expressions of these two proteins were signi cantly upregulated 7 days after UUO surgery.It is noteworthy that their expressions at 14 days were signi cantly higher than those at 7 days and in the sham groups (Fig. 1E, 1F).These ndings indicate that SCF/c-Kit may be involved in renal interstitial brosis caused by UUO surgery.

c-Kit gene mutation facilitates renal function recovery after UUO surgery
In order to further understand the role of SCF/c-Kit in renal brosis, homozygous c-Kit mutant Wps/Wps mouse strain was used in the subsequent experiments.Wps/Wps mouse strain is characterized by lossof-function mutation in the c-Kit gene.No obvious difference of pathological staining and renal function were observed between Wps/Wps mice and wildtype mice of the same age (Fig. 2A, 2B).The degree of increase in serum creatinine level in Wps/Wps 7 days after UUO surgery was signi cantly lower than that in wildtype mice (1.18 times and 1.82 times the baseline level, respectively).This was also the case with respect to the BUN level, which was 1.28 times and 2.14 times the baseline value, respectively.The serum creatinine and BUN levels of Wps/Wps mice almost returned to baseline levels 14 days after UUO surgery (Fig. 2B-2D).These results indicate that de ciency of c-Kit facilitates renal function recovery after UUO surgery.

c-Kit gene mutation alleviates kidney tissue damage, interstitial brosis, and NF-κB phosphorylation in UUO model
Renal tubular epithelial cell swelling, necrosis and shedding, formation of casts, interstitial edema, and brosis were observed in the WTuuo14 group.Compared to the WTuuo14 group, the kidney damage and the brosis areas were signi cantly reduced in the Wps/Wpsuuo14 group (Fig. 2E, 2F), which suggests that c-Kit gene mutation may alleviate UUO-induced kidney tissue damage and renal interstitial brosis.
The expressions of brosis-related proteins such as ColI, α-SMA, and TGF-β1 in the WTuuo14 group were signi cantly greater than that in the control and the Wps/Wpsuuo14 groups (Fig. 3A-3D).The immuno uorescence results indicated that the α-SMA-and TGF-β1-positive cell ratios in the Wps/Wpsuuo14 group were signi cantly lower than that in wildtype mice (Fig. 3F-3I), which further indicates that c-Kit de ciency contributed to the alleviation of UUO-induced renal interstitial brosis.The ratio of phosphor-NF-κB/total NF-κB was upregulated after UUO surgery.Moreover, the elevated level of the ratio in the Wps/Wpsuuo14 group was signi cantly lower than that in the WTuuo14 group (Fig. 3A,  3E), which suggests that SCF/c-Kit may activate NF-κB.

Youthful blood environment may alleviate elderly mice kidney brosis by reducing NF-κB activation
The expressions of SCF, c-Kit, TGF-β1, and the phosphor-NF-κB/total NF-κB ratio in the Ouuo14 group were signi cantly higher than those in the Yuuo14 group (p < 0.01).Compared to the Ouuo14 and O-IPuuo14 groups, their expressions in the O-HPuuo14 group decreased signi cantly (p < 0.01).The expressions of SCF and TGF-β1 in the O-HPuuo14 group were similar to those in the Y-IPuuo14 group (p > 0.05) (Figure S3A-S3E).The phosphor-NF-κB/total NF-κB ratio in the O-HPuuo14 group was remarkably lower than that in the Ouuo14 and O-IPuuo14 groups.These results suggest that youthful blood environment downregulates the expression of SCF/c-Kit in elderly UUO mice and inhibits renal brosis, and that these effects may be mediated via the NF-κB pathway.

Discussion
In our previous study, we have identi ed that youthful blood environment can reduce the extent of agerelated increase in kidney brosis area using parabiotic mice model [9].However, when the elderly rat kidney was transplanted into a young rat, no obvious change in kidney brosis of the elderly rat was observed [10].In this study, we further sought to clarify the effect of blood environmental change on senile renal interstitial brosis.We established the UUO model using parabiotic mice.UUO surgery was performed 3 weeks after parabiosis.One reason is that the stable common circulation is formed usually 2 weeks after parabiotic surgery.In addition, the death rate is high in the rst 2-3 weeks after parabiotic surgery.Therefore, by selecting parabiotic mice 3 weeks after parabiosis, the failure of modeling caused by 'parabiosis disease' can be avoided to save time and costs.The success rate of the parabiotic mice model combined with UUO surgery was 80-90%, which laid the foundation for further research.
Compared with the young individuals, older individuals have poor tolerance for various types of trauma, and it is not easy to recover after more serious injury [22].It has been shown that a signi cantly higher proportion of elderly patients progress to end-stage renal disease after acute kidney injury or unilateral ureteral obstruction as compared to young patients [23].In this study, the levels of serum creatinine and BUN in young mice reached peak levels at 7-10 days after UUO surgery, and subsequently recovered to varying degrees at 14 days after surgery; these changes may be associated with the compensatory effect of the contralateral kidney [24].However, in case of elderly mice, the renal function continued to deteriorate until 14 days after surgery, which suggests decreased compensatory function and lower ability to repair damaged kidney of elderly mice.The renal function of O-HPuuo14 mice did not get worse or even improved, which suggests that youthful blood environment can reduce the renal injury caused by UUO, and promote renal function recovery after damage.
The pathological process of renal brosis can be divided into the following phases [15]: (1) start-up phase: cell activation and damage; (2) activation phase: activation of brosis signaling pathway; (3) brosis stage: extracellular matrix accumulation period; (4) brosis progression phase.Renal blood ow and glomerular ltration rate were shown to decrease within 24 hours of complete unilateral ureteral obstruction, followed by development of hydronephrosis, tubulo-interstitial in ammatory in ltration, and kidney tubular cell apoptosis or necrosis [25].Severe hydronephrosis and atrophy of renal parenchyma typically occurs 1-2 weeks after UUO surgery [26].In this study, the UUO-induced kidney tissue injury was more severe in elderly mice compared to young mice, and was accompanied with severe renal interstitial brosis at 2 weeks after surgery.The extent of kidney damage or brosis in the O-HPuuo mice was lower than that in the Ouuo or O-IPuuo mice.The difference was especially obvious at 2 weeks after surgery, i.e., around the time of the formation and accumulation of extracellular matrix [27].This indicates that some 'anti-brosis' factors may exist in youthful blood environment which play a role in inhibiting kidney brosis after UUO.Studies have shown that multiple signaling pathways are involved in renal brosis process, including the TGF-β/Smad [20,28], Wnt/beta-catenin [29], and p38MAPK [30] pathways.Moreover, the sharp decline in renal blood ow and glomerular ltration rate after UUO surgery and the elevated angiotensin II level promote secretion of TGF-β1 by macrophages, which triggers the proliferation of broblasts and their differentiation to myo broblasts; the myo broblasts secrete collagen that is helpful for renal tubular interstitial brosis [31].High expression of TGF-β1 has been shown to be closely related to renal brosis [32][33][34].Our research shows that youthful blood environment can signi cantly reduce the expression of TGF-β1 in elderly renal tissue after UUO, and can also reduce the protein expressions of myo broblast surface markers α-SMA and collagen ColI.
In previous study, we compared the serum cytokine levels in young and elderly mice with 5 weeks of IP or HP, and found that stem cell factor (SCF) may be associated with renal senescence; in addition, we con rmed higher expression of SCF in blood of elderly mice.Several studies have shown high expression levels of SCF and its receptor c-Kit in renal brosis tissue [18][19][20].In this study, the expressions of SCF and c-Kit in kidney tissue of elderly mice were signi cantly higher than those in young mice.The expressions of SCF and c-Kit in renal tissue after UUO were signi cantly higher than those in the sham group, and continued to increase with increase in the duration of renal obstruction; these ndings suggest that SCF/c-Kit are important for UUO-induced renal tissue damage.We further used loss-of-function c-Kit gene mutant mice Wps/Wps strain to construct UUO model, and the mutant mice showed lower degree of kidney brosis, lower levels of serum creatinine and BUN, and decreased expressions of key brosisrelated cytokines TGF-β, and myo broblast surface markers α-SMA and collagen ColI, compared to wildtype mice.In addition, the serum creatinine and BUN levels almost recovered to baseline level 2 weeks after UUO.These results further con rmed the pro-brogenic effects of SCF/c-Kit during renal tubular interstitial brosis.Blockade of the SCF/c-Kit signaling pathway can alleviate UUO-induced renal interstitial brosis and promote renal function recovery after UUO injury.
Binding of SCF to its receptor c-Kit has been shown to promote the expressions of a variety of in ammatory cytokines, including histamine, tryptase, transforming growth factor (TGF-β), broblast growth factor (FGF-β), and NF-κB [35,36].Studies have shown that SCF/c-Kit can mediate airway in ammation through activation of the NF-κB signaling pathway [37], regulate tumor cell in ammatory microenvironment [38], promote pulmonary interstitial brosis [39], and remodel the vascular neural network in chronic stroke patients [40].In a fundamental research about spontaneous hypertension, high expressions of SCF/c-Kit were shown to activate NF-κB expression, and promote myocardial hypertrophy and myocardial brosis [41].In this study, more severe UUO-induced injury was observed in elderly mice with high expressions of SCF/c-Kit and NF-κB, compared to young mice.The youthful blood environment in O-HPuuo mice downregulated the expressions of SCF/c-Kit and NF-κB, and the extent of kidney damage in O-HPuuo14 was similar to that in the young mice group.Moreover, the c-Kit loss-of-function mutant Wps/Wps mice exhibited decreased expression of NF-κB and reduced extent of kidney interstitial brosis.These results indicate that youthful blood environment downregulates the high expression of SCF/c-Kit in elderly kidney tissue, and subsequently inhibits NF-κB expression and decreases elderly renal interstitial brosis.

Conclusions
We established a combination mice model of unilateral ureteral obstructive (UUO) and parabiotic surgeries, and found that youthful blood environment can signi cantly reduce UUO-induced renal interstitial brosis in elderly mice, and promote kidney function recovery by downregulating the expression of SCF/c-Kit in elderly kidney tissue.Using Wps/Wps mice with loss-of-function mutation in SCF receptor c-Kit, we demonstrated for the rst time the role of SCF/c-Kit in the process of senile renal brosis, which may be mediated via the NF-κB pathway.These results provide the basic experimental evidence for prevention and treatment of renal interstitial brosis, which may help formulate strategies to attenuate kidney disease progression and age-related renal damage.Western Blot analysis showed both of the proteins in elderly mice kidney tissues were signi cantly higher than those in young mice.(D) Graph representing quantitative analysis results of SCF and c-Kit.(E) The expressions of SCF and c-Kit at different time after surgery.Western Blot analysis showed 14 days after UUO surgery, both of proteins were signi cantly higher than those at 7 days and in the sham groups.(F) Graph representing quantitative analysis results of SCF and c-Kit.UUO: unilateral ureteral obstructive; UUO7d: 7 days after UUO surgery; UUO14d: 14 days after UUO surgery.The expressions of phosphor-NF-κB and brosis-related proteins ColI, α-SMA, and TGF-β1 in the four groups.Western Blot analysis showed 14 days after UUO surgery, all these brosis-related proteins and the ratio of phosphor-NF-κB/total NF-κB in WT groups were signi cantly higher than those in C-KIT-/groups.(B-E) Graph representing quantitative analysis results of these four proteins.(F) Abbreviations stem cell factor;

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