Effect Of Phenylethanol Glycosides On The Transforming Growth Factor – P1/Smads Signaling Pathway in The Human Hypertrophic Scar Fibroblasts

Effect of phenylethanoid glycosides on the transforming growth factor – p1/Smads signaling pathway in the human hypertrophic scar fibroblasts

For more information:Ali.ma@wecistanche.com

[Abstract] Objective Based on the transforming growth factor - pl (TGF - pl)/Smads signaling pathway to investigate the effects of phenylethanoid glycosides from cistanche tubulosa (CPhGs) on human hy­pertrophic scar fibroblasts (HSFbs) and the possible mechanism. Methods From March to June 2019, 9 patients with hypertrophic scar after bum were admitted. The fibroblasts were isolated from hypertrophic scar and cultured. Human hypertrophic scars were removed and fibroblasts were cultured in vitro. The blank group, model group, positive control group (TGF - pl + 5 - fluorouracil) , and experimental group (TGF - pl + CPhGs) were set up. The effects of phenylethanoid glycosides from cistanche tubulosa at different concentrations on the proliferation of HSFbs were detected by the cell counting kit - 8 (CCK - 8) assay. The expression of Smad2, Smad3, Smad7, collagen I (COL -1) and collagen HI (COL -3) mRNA was detected by real - time quantita­tive reverse transcriptase - polymerase chain reaction (RT - qPCR) in each group. The expression of Smad2, Smad3, phosphorylated Smad2 (p - Smad2), phosphorylated Smad3 (p - Smad3), Smad7, COL -1 and COL - 3 proteins was detected by Western blotting. Measurement data were expressed as mean ± standard deviation (Mean ± SD). Multiple groups were compared using one - way ANOVA. Results The inhibition rate of fibroblasts after treatment with CPhGs at 60 , 90, and 120 mg/L for 48 h was (36.87 ±0.06)% vs. (43.52 ±0.04)% vs. (78.11 ±0.03)% , F value =45.070, P<0.01. The inhibitory effects of CPhGs on fibroblasts increased with the increase of drug concentration and the prolonga­tion of action time. RT - qPCR results showed that as compared with the model group, the expression of COL -1, COL - 3, Smad2, Smad3 mRNA was reduced, and the expression of Smad7 mRNA was in­creased in the experimental and positive control groups (0.21 ± 0.04 vs. 0.3 ±0.25 vs. 1.56 ± 0.01, F value =39.755, P<0.01), (0.55 ±0.04 vs. 0.27 ±0.04 vs. 1.66 ±0.22, F value = 61.991, P< 0.01), (0.55 ±0.03 vs. 0.78 ±0.76 vs. 1.06 ±0.15, F value = 17.769, P<0.01), (0.46 ±0.08 vs. 0.55 ± 0.10 vs. 2.05 ± 0.29, F value = 56.796, P < 0.01), (1.77 ± 0.59 vs. 0.11 ± 0.02 vs.

0.04 ± 0.04, F value = 14.780, P < 0.05) ； Western blotting results showed that the protein expression of p-Smad2 and p -Smad3, COL - 1, COL -3, in the experimental group and positive control group was significantly lower than that in the model group, and the expression of Smad7 protein was significantly high­er than that in the model group (0. 16 ± 0.03 vs. 0. 11 ± 0.02 vs. 0.06 ± 0.00), (0. 12 ± 0.02 vs.

0.07 ± 0.01 vs. 0.04 ±0.01, F value =7.670, P < 0.05), (0.12 ±0.03 vs. 0.15 ±0.06 vs. 0.23 土 0.05, F value =3.936, P<0.05), (0.27 ±0.02 vs. 0.08 ±0.01 vs. 0.05 ±0.04, F value = 34.291, P < 0.01), (0.33 ±0.02 vs. 0.16 ±0.02 vs. 0.17 ±0.01 F value = 16.322, P<0.01). Conclusion CPhG inhibited hypertrophic scar fibroblasts proliferation and activation via blocking the TGF - pl/Smad signaling.

[Key words] Phenylethanoid glycosides from cistanche tubulosa； Hypertrophic scar fibroblasts； Transforming growth factor - pl/Smads signaling pathway

Click to cistanche deserticola ma and Cistanche products

Hypertrophic scarring (HS) is a fibrotic disease of the skin characterized by excessive proliferation of fibroblasts (FBS), as well as extracellular matrix (ECM) deposition, which are the main enabling cells for scar formation, can secrete large amounts of collagen, and the transforming growth factor (TGF) - P / Smads signaling pathway is closely related to the formation of HS. Cistanche is a traditional tonic traditional Chinese medicine, the extract of which phenylethanoid acetic acid total (cphgs) is one of the main ingredients exerting medicinal effects and having various biological functions such as anti-inflammation, anti-tumor, anti-radiation, free radical scavenging, etc. studies have shown that cphgs can inhibit hepatic stellate cell proliferation, and that it can do so by decreasing the expression of Smad2, Smad3, Increasing the expression of Smad7 thereby blocked tgf-p / Smads signaling pathway transduction and then delayed the progression of liver fibrosis. The aim of this study was to observe the effects on the expression of genes and proteins related to human hypertrophic scar fibroblasts (hsfbs) based on the TGF PL / Smad signaling pathway.

Materials and methods

1. Materials: Cistanche cphgs were purchased from hechen pharmaceutical biotech Co., Ltd., Hetian, China; Fetal bovine serum was purchased from Clark Bioscience, USA; Cell counting kit TGF PL was purchased from pepro tech, USA; The fluorescence quantitative PCR kit was purchased from Takara Corporation, Japan; Reverse transcription kits were all purchased from Thermo Fisher Scientific, USA; Smad2 / 3, p-Smad2 / 3 antibodies were purchased from cell signaling technology, USA; Antibodies to Smad7, COL-1, COL-3, glyceraldehyde-3-phosphate dehydrogenase (gap-dh) were purchased from abeam (UK); Polyvinylidene fluoride (PVDF) membranes were purchased from millipore, USA.

2. The cases were all selected from the Department of orthopedics, the First Affiliated Hospital of Xinjiang Medical University, and three specimens (all of which were definitely diagnosed as hypertrophic scar by pathology) were randomly selected for primary culture of FBS; This experiment was approved by the medical ethics committee of the First Affiliated Hospital of Xinjiang Medical University (approval number 20170214-71), and informed consent was obtained from the patients and their families.

3. Cell grouping: randomly divided into blank group; Model group: 5 |ug / L TGF - & 1; Positive control: 5|ug / L TGF PI + 250 mg / L 5-fluorouracil (5-FU); Experimental group: 5|ug / L TGF PL + (60 mg / L, 90 mg / L >, 120 mg / L) cphgso.

4. Cell proliferation was determined by cell counting Kit (CCK-8): hsfbs in logarithmic growth phase were taken, cells from each group were treated as designed, and the absorbance value of each well of each group was monitored sequentially at 450 nm wavelength using a microplate reader at 24, 48 and 72 hours.

5. Detection of mRNA expression by RT qPCR: hsfbs from each group after 48 hours of intervention were collected, RNA was extracted and the amount of mRNA expression was determined.

6. Protein expression was determined by Western blot analysis. Total cellular protein was extracted 48 hours after treatment from each group to detect the expression of each protein.

7. Statistical methods: the statistical software SPSS 22.0 was used to analyze, and the measurement data were expressed as mean ± standard deviation (mean ± SD), and one-way analysis of variance was used for comparison among multiple groups, and P < 0.05 was considered statistically significant.

Results

Cell proliferation results showed that 60, 90, and 120 mg / L cphgs for 48 hours on fibroblasts, the inhibition rate was [(36.87 ±0.06)%: (43.52 ±0.04)% :(78.11 ±0.03)% ,F=45.073,P< 0.01 ],90 mg/L CPhGs. 24h-28h was [(26.29 ±0.04)% :(43.52 ±0.04)% :(64.11 土 0.03)%,F = 59.770,P<0.01]. It indicated that the inhibition rate of cphgs on hsfbs was gradually elevated with time extension and increasing concentration, showing a proportional relationship.

Comparison of CoL-I, COL-3, Smad2, and Smad3 mRNA expression in each group: compared with the model group, both the positive control and experimental groups could reduce transforming growth factor β - induced CoL-I, COL-3, Smad2, and Smad3 mRNA expression, and promote Smad7 mRNA expression, with statistically significant differences. (1.56 ±0.01:0. 30 ± 0.25 : 0.21 ±0.04,F = 39. 755,P <0.01； 1.66 ±0. 22 : 0. 27 ±0.04 : 0. 55 ±0. 04.F = 61. 991 ,P <0. 01； 1. 06 ± 0.15 比 0. 78 ± 0. 76 比 0. 55 ± 0. 03, F = 17.769 ,P <0.01 ；2.05 ± 0.29 : 0.55 ± 0.10 : 0.46± 0.08,F = 56.796,P<0.01 ；0.04 ±0.04 : 0.11 ±0.02 : 1.77±0. 59,F = 14. 780,P<0. 05)。

Comparison of p-Smad2, p-smad3, col-l, COL-3 in each group: compared with the blank group, the protein expression of p-Smad2, col-l in the model group increased, while that of Smad7 decreased; Compared with the model group, the positive control group could downregulate the protein expression of p-smad3, COL-1, COL-3, and the experimental group showed an inhibitory effect on the elevated protein expression of p-Smad2, p-smad3, col - 3. COL-3 expression was inhibited to various degrees, whereas the expression of Smad7 was relatively increased in the two groups, and the protein expression values of p-Smad2, p-smad3, Smad7, col-l, COL-3 in the blank group, the positive control group, and the experimental group were (0.09 ±0.04,0.16 ±0.03,0.11 ± 0.02,0.06 ± 0.00),(0.07 ±0.03,0.12 ± 0.02,0.07 ± 0.01,0. 04 ±0. 01,F = 7. 670,P <0. 05), (0. 22 ±0.05、 0.12 ±0.03,0.15 ±0.06,0.23 ± 0.05, F= 3.936, P< 0.05),(0.19 ±0.02,0.27 ± 0.02,0.08 ±0.01,0.05 ± 0.04,F = 34.291,P<0.01)、(0.21 ±0.04.0. 33 ±0.02、 0.16 ±0.02,0.17 ±0.01,F = 16.322,P<0.01),

Discussion

HS is an inevitable outcome of tissue repair, and its occurrence mechanism mainly FBS hyperproliferation, insufficient degradation and so on lead to the formation and development of HS. Tgf-p1 is widely involved in cellular, tissue abnormal proliferation and differentiation diseases and continues throughout wound healing; It exerts its effects mainly through the tgf-p / Smads signaling pathway, in which phosphorylation of Smad2 / 3 proteins leads to increased expression of intracellular and extracellular fibro genic proteins, promotes collagen production, and accelerates fibrosis progression; Smad7, as a negative regulator, may prevent this pathway through interaction with activated tgf-p1 receptors, thereby reducing collagen fiber synthesis; Thus either increasing Smad7 expression or inhibiting Smad2 / 3 expression can prevent hypertrophic scar development.

This study applied Cistanche cphgs to HS, a skin fibrotic disease, and found that various doses of cphgs inhibited the proliferation of hsfbs induced by tgf-p1; Moreover, 90m & / L cphgs inhibited pathway signaling by promoting Smad7 expression and reducing Smad2 / 3 expression at both gene and protein expression levels, and reduced CoL-I, COL-3 production, which in turn inhibited HS progression. Our results confirmed that cphgs inhibited collagen production by intervening in the expression of proteins related to tgf-p / Smads signaling pathway under tgf-p1 stimulation. The results of the present study demonstrated that Cistanche cphgs inhibited the proliferation and effectively reduced collagen secretion of hypertrophic scar fibroblasts through the tgf-p / Smads signaling pathway.