Parishin alleviates vascular ageing in mice by upregulation of Klotho

Abstract

Senescence of vascular endothelial cells is the major risk of vascular dysfunction and disease among elderly people. Parishin, which is a phenolic glucoside derived from Gastrodia elata, significantly prolonged yeast lifespan. However, the action of parishin in vascular ageing remains poorly understood. Here, we treated human coronary artery endothelial cells (HCAEC) and naturally aged mice by parishin. Parishin alleviated HCAEC senescence and general age-related features in vascular tissue in naturally aged mice. Network pharmacology approach was applied to determine the compound-target networks of parishin. Our analysis indicated that parishin had a strong binding affinity for Klotho. Expression of Klotho, a protein of age-related declines, was upregulated by parishin in serum and vascular tissue in naturally aged mice. Furthermore, FoxO1, on Klotho/FoxO1 signalling pathway, was increased in the parishin-intervened group, accompanied by the downregulated phosphorylated FoxO1. Taken together, parishin can increase Klotho expression to alleviate vascular endothelial cell senescence and vascular ageing.

Keywords: FoxO1; Klotho; ageing; network pharmacology; parishin; senescence; vascular endotheliocyte.

FIGURE 1

Parishin delays HCAEC replicative senescence. (A) Immunofluorescence staining of γH2AX expression in the high‐dose parishin intervention groups under the indicated conditions. Scale bar, 10 μm. (B) Western blot was used to examine the expression of PARP 1 γH2AX, IL‐6 and p16^Ink4a protein expression in replicative senescent cells among different groups. (C) Differences in mitochondrial morphology between the high‐dose parishin intervention group and the elderly control group. (D) Differences in total ROS expression between the high‐dose parishin intervention group and the elderly control group. (E) The corresponding quantitative data of ROS expression. **p < 0.01, versus the control group.

FIGURE 2

Parishin reduces senescent markers of vascular tissues and serum in naturally aged mice. (A) Temporal schematic diagram of the experimental procedures. (B, C) Western blotting and quantification of lamin B1and IL‐6 in vascular tissue of different groups. (D) The degree of vascular fibrosis was investigated by Masson's trichrome staining (magnification 40×). (E) The corresponding quantitative data of Masson's trichrome staining. (F) Serum GDF15, CXCL9 and sFlt1 levels in different groups were measured using ELISA kits. *p < 0.05 versus Control mice.

FIGURE 3

Parishin improves endothelial cell function in naturally aged mice. (A) Immunohistochemistry results revealed the expression of PPARα in different groups (magnification 20×). (B) The corresponding quantitative data analysis of PPARα expression was done with ImageJ software. (C, D) Western blot was used to examine the expression of PPARα protein in each vascular tissue. (E) Immunohistochemistry results revealed the expression of eNOS in different groups (magnification 40×). (F) The corresponding quantitative data analysis of eNOS expression was done with ImageJ software. ** p < 0.01, versus the control group. * p < 0.05, versus the control group.

FIGURE 4

Parishin increases α‐Klotho of vascular tissue in normal ageing mice. (A) The human serum α‐Klotho levels among the Young and Old groups were measured using ELISA. Pearson's correlation analysis of α‐Klotho levels based on age. (B) Serum α‐Klotho levels in different ageing mice groups were measured using ELISA kits. (C) Western blot was used to examine the expression of α‐Klotho protein in each vascular tissue. (D) Immunohistochemistry results revealed the expression of α‐Klotho in different groups (magnification 20×). (E) The corresponding quantitative data analysis of α‐Klotho expression was done with ImageJ software. (F) Immunofluorescence staining of α‐Klotho expression in the high‐dose parishin intervention groups under the indicated conditions. Scale bar, 20 μm. (G) The corresponding quantitative data analysis of α‐Klotho expression was done with ImageJ software. ***p < 0.001, versus the control group. **p < 0.01, versus the control group. *p < 0.05, versus the control group.

FIGURE 5

Parishin upregulates FoxO1 in naturally aged mice. (A) Immunohistochemistry results revealed the expression of FoxO1 in different groups (magnification 20×). (B) The corresponding quantitative data analysis of FoxO1 expression was done with ImageJ software. (C) Immunofluorescence staining of FoxO1 expression in different groups, Scale bar, 20 μm. (D, E) Western blot was used to examine the expression of FoxO1 and P‐FoxO1 protein in each vascular tissue. ****p < 0.001, versus the control group. *p < 0.05, versus the control group.

FIGURE 6

Summary of the molecular mechanism of the anti‐ageing effect of parishin. Parishin treatment reduced the production of ROS, further improved mitochondrial function, then activated the Klotho/FoxO1 signalling pathway and finally exerted the anti‐ageing effect.