Effects Of Saponin From The Seed Of Litchi Chinensis Sonn On TGF-β1, FN And SOCS-1 in Renal Tubular Epithelial Cells Under High Glucose

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Hai-Yang Nie, Rui Chen, Hong-Na Zhang, Zhi Pan

1 Changchun University of Chinese Medicine, Changchun, China.

2 Shunyi Hospital of Beijing Chinese Medicine Hospital, Beijing, China.

Highlights

Saponin from the seed of Litchi chinensis Sonn reduces the apoptosis of renal tubular epithelial cells and the secretion of TGF-β1 and FN.

Abstract

Objective: To investigate the effect of saponin from the seed of Litchi chinensis Sonn (SLS) on the growth and apoptosis of human kidney epithelial cells (HKC) cultured in high glucose. Methods: HKC were cultured in DMEM/F12 medium supplemented with 30 mmol/L glucose and treated with or without SLS. In the normal group, an isometric DMEM/F12 medium with 5.5mmol/L glucose was added. The secretion of TGF-β1 and fibronectin (FN) were detected by ELISA. Cell apoptosis was detected by the method of Annexin V-FITC/PI double staining. Western blot was used to detect the level of suppressor of cytokine signaling-1 (SOCS-1).

Results: The result of ELISA showed that the secretion of TGF-β1 and FN was decreased in SLS groups compared with those in 30 mmol/L glucose-treated groups (P < 0.05). There were more cells apoptosis in the 30 mmol/L glucose treated group than that in the normal group (P < 0.01). Compared with the 30 mmol/L glucose treated group, the apoptosis of HKC was significantly decreased in SLS groups (P < 0.01). Western blot showed that the level of SOCS-1 in the high glucose + SLS group was decreased (P < 0.01), compared with the high glucose group.

Conclusion: SLS can reduce the secretion of TGF-β1 and FN in HKC by reducing the deposition of the extracellular matrix. SLS also significantly reduced the apoptosis of HKC by inhibiting the level of SOCS-1. These results suggest the roles of SLS in preventing the progress of glomerular sclerosis.

Keywords: Saponin, Seed of Litchi chinensis Sonn, Glucose, Kidney epithelial cells, TGF-β1, Fibronectin, Apoptosis

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Background

Diabetic nephropathy (DN)is one of the most serious complications of diabetes mellitus with high incidence and lethality, characterized by glomerular sclerosis and renal interstitial fibrosis. Human kidney epithelial cells (HKC)are the major targets in DN and they secret a large number of cytokines, such as TGF-β1 and fibronectin (FN), which lead to the occurrence of glomerulosclerosis and renal interstitial fibrosis. Meanwhile, it has been reported that high glucose can accelerate HKC apoptosis by increasing the level of suppressor of cytokine signaling-1(SOCS-1)[1,2]. Therefore, inhibiting the level of TGF-β1, FN and SOCS-1 is important for preventing the progress of glomerular sclerosis. The seed of Litchi chinensis Somm(SLS), is a Chinese herb mainly distributed in Guangdong. Fujian and Guangxi provinces of China were first recorded in Bencaogangmu in the Ming Dynasty of China(1518 A.D.-1593 A.D.). Saponin is the active ingredient of the seed of Litchi chinensis Sony. Based on our previous study, this study further explores the effect of SLS on the level of TGF-β1 and FN in HKC cultured with high glucose, the apoptosis of HKC, and the underlying mechanism on the prevention and therapy of glomerulosclerosis [3].

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Materials and methods Materials

SLS with a purity of more than 98% was purchased from Xianqingle Biological Co., LTD (Xi'an, China).HKC was purchased from Shanghai Fuxiang Biotechnology Co., Ltd(Shanghai, China).DMEM/F12 medium, standard fetal bovine serum, and trypsin were from Hyclone(USA). The dimethyl subfamily was from Sigma (USA). ELISA kit was purchased from Shanghai Longton Biotechnology Company(Shanghai, China). Annexin V-FITC/PI Apoptosis Detection Kit was from Nanjing Kaiji Biotechnology Co., Ltd (Nanjing, China).

Cells culture

HKC were maintained in DMEM/F12 with 10% fetal calf serum and were cultured at 37℃ in a humidified atmosphere of 95% air and 5% CO.Cells were digested with 0.25% trypsin after 2-3 days for use.

Enzyme-linked immunosorbent assay

HKC were divided into six groups, including control group(DMEM with 5.5mmo/L glucose),high glucose group (DMEM with 30mmol/L glucose), SLS low dose group(5mg/ml, S1+30mmol/L glucose),SLS medium dose group (10mg/ml, S2+30mmol/L glucose), SLS high dose group (20mg /ml, S3+30mmol/Lglucose),and blank tone group (DMEM without cells).

48 hours later, the supernatant of each group was collected, and the content of TGF-β1 and FN were detected by ELISA. The operation was carried out according to the manual. The OD value was read at 450nm by a microplate reader. The concentrations of TGF-β1 and FN were determined according to the standard curve.

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Flow cytometry

The experimental groups were the same as above. The apoptosis cells were detected by flow cytometry with Annexin V-FITC/PI double staining. The upper left quadrant represented the percentage of necrotic cells, the upper right quadrant represented the percentage of late apoptosis cells, the lower left quadrant represented the percentage of viable cells, and the lower right quadrant represented the percentage of early apoptosis cells.

Western blot analysis

HKC was treated with 10* mol/L of Angiotension (Ang)Ⅱ,20mg/Lof SLS and 10*mol/L ofAngⅡ, DMEM/F12 medium, 20mg/L of SLS and 25mmol/L of glucose and 25mmol/L of glucose, respectively.

Cell lysate proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Each membrane was incubated with a monoclonal antibody, followed by incubation with peroxidase-conjugated secondary antibodies and chemiluminescence detection.

Statistical analysis

All statistical analyses were performed using SPSS version 19.0. The difference between the experimental data groups was analyzed by one-way ANOVA, and the least significant difference was used between the groups. P<0.05 was considered to indicate statistical significance.

Results

Effects of SLS on the secretion of TGF-B1 and FN Compared with the high glucose group, SLS could inhibit the levels of TGF-B1 and FN in HKC (P<0.01 or P<0.05)(Figure 1, Figure 2).

Compared with the high glucose group: * P<0.05, ** P<0.01. Control group: DMEM with 5.5mmol/L glucose, high glucose group: DMEM with 30mmol/L glucose, S1 group: 5mg/ml SLS+30mmol/L glucose, S2 group: 10mg/ml SLS+30mmol/L glucose, S3 group: 20mg /ml SLS+30mmol/L glucose. SLS, Seed of Litchi chinensis Sonn.

Effect of SLS on HKC apoptosis The result of flow cytometry showed that the apoptosis of the high glucose group was significantly higher than the normal group (P < 0.01). Compared with the high glucose group, the apoptosis rate of HKC was negatively correlated with concentrations of SLS (P < 0.01) (Figure 3).

Effect of SLS on the level of SOCS-1 The level of SOCS-1 protein in the AngII group was significantly higher than that in the normal group (P < 0.01). The level of SOCS-1 in the AngII+SLS group was lower than that in the AngII group but still higher than that in the control group (P < 0.01). Compared with the normal group, the level of SOCS-1 protein in the high glucose group was increased (P < 0.01). Compared with the high glucose group, the protein level of the high glucose+SLS group was decreased (P < 0.01) (Figure 4).

Figure 4 Effect of SLS on the expression of SOCS-1 a. AngII group: 10-6 mol/L Angiotension Ⅱ, b. AngII+SLS group: 20mg/L SLS and 10-6 mol/L AngⅡ, c. Control group: DMEM/F12 medium, d. High glucose+SLS group: 20mg/L SLS+25mmol/L of glucose, e. Control group: DMEM/F12 medium, f. High glucose group: 25mmol/L glucose. SOCS-1, suppressor of cytokine signaling-1. SLS, Seed of Litchi chinensis Sonn.

Discussion

Deposition of extracellular matrix and apoptosis of renal tubular epithelium is the main pathological changes in the development of DN. Under normal circumstances, the morphology and number of HKC remained relatively stable. But in a variety of stimulating factors such as inflammation, HKC can significantly proliferate [4]. High glucose leads to the high secretion of TGF-β1 and FN of HKC and accelerates the apoptosis of HKC, which is a direct factor in promoting the occurrence of DN. In this experiment, high glucose was used as a stimulus. It was found that SLS could inhibit the secretion of TGF-β1, FN and reduce the HKC apoptosis by decreasing the level of SOCS-1.

Litchi chinensis Sonn is mainly distributed in Guangdong, Fujian, and Guangxi provinces of China. It was reported that the SLS possessed antihyperglycemic, antihyperlipidemic, antiplatelet, and antiviral activities [5]. According to the record of Bencaogangmu in the Ming Dynasty of China (1518 A.D. - 1593 A.D.), the SLS can “relieve thirstily”. In recent years, many scholars have done a lot of research on its pharmacological effects and chemical composition. The main chemical components of the SLS are compounds such as lychee saponins, flavonoids, polyphenols, chisels, amino acids, fatty acids, and volatile components. SLS is the main active ingredient. The SLS is widely used in diabetes and diabetic nephropathy, with hypoglycemic, hypolipidemic, inhibition of immune inflammation effects [6]. Modern experimental studies also have confirmed that SLS has a hypoglycemic effect [7]. However, the mechanism of this effect is not clear.

In this study, the level of TGF-β1 and FN in HKC was increased significantly after stimulation with high glucose, and the level of TGF-β1 and FN were decreased significantly after SLS treatment in a dose-dependent manner. SOCS-1 protein level was decreased significantly after SLS treatment. This study was provided a theoretical basis for the clinical application of SLS.

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References

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