Efficacy of Dangua Fang (丹瓜方) on endothelial cells damaged by oxidative stress

doi:10.19852/j.cnki.jtcm.20220815.001

Abstract

Abstract:

OBJECTIVE: To evaluate the protective effects of serum containing Dangua Fang (丹瓜方) on vascular endothelium damaged by oxidative stress.

METHODS: Five experiments were completed in this paper. In the first experiment, we found the most suitable serum containing Dangua Fang by comparing groups with different serum containing Dangua Fang. In the second experiments we analyzed Dangua Fang influencing endothelial cell viability and apoptosis and cell cycle. The third experiment on Dangua Fang intervention of mitochondrial respiratory chain. The fourth experiment on Dangua Fang intervention of mitochondrial membrane potential. And finally, on the fifth experiment we researched the mechanism of Dangua Fang improving mitochondrial function by comparing the Na⁺-k⁺-ATPase and peroxisome proliferator-activated receptor- gamma coactivator-1alpha (PGC-1α) in the Dangua group with the diazoxide group and Co Q+Vit C group.

RESULTS: We compared the control group in the first experiments and the OD values in DZ1 group was the most significant in all intervening groups. The recipe of DZ1 (5% serum containing Dangua Fang) was used in the following experiments. Compared with the control group, cell viability, cell cycle (G2 + S), cytochrome c oxidase (COX), R3 red/green, R2 red/green, R1 red/ green decreased and apoptosis, succinate dehy-drogenase (SDH), green (R2 + R3), Na⁺-k⁺-ATPase, PGC-1α increased in the model group. Compared with the model group, cell viability, G2+S, COX, R3 red/green, R2 red/green, R1 red/green raised and apoptosis, green (R2 + R3), Na-K-ATPase decreased in the Dangua group; G2 + S, R3 red/green, R2 red/green, R1 red/green raised and green (R2 + R3) decreased in the Co Q + Vit C group. Na-K-ATPase increased in the combined group (P < 0.05 or < 0.01).

CONCLUSIONS: Dangua Fang protects oxidative stress-induced endothelial cells damaged by promotion of mitochondrial biogenesis, reduction of Na⁺-K⁺-ATPase activity and regulation of mitochondrial respiratory chain function restoring mitochondrial membrane potential.

Key words: mitochondria, oxidative stress, endothelial cells, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, Dangua Fang

HENG Xianpei, LI Liang, YANG Liuqin, WANG Zhita. Efficacy of Dangua Fang (丹瓜方) on endothelial cells damaged by oxidative stress[J]. Journal of Traditional Chinese Medicine, 2022, 42(6): 900-907.

Figures/Tables 6

Table 1 Comparison of OD values of different concentrations of Dangua Fang-containing serum intervention of endothelial cell proliferation activity injure induced by oxidative stress ($\bar{x} \pm s$)

Group	n	OD value	ReR (%)	Group	n	OD value	ReR (%)	Group	n	OD value	ReR (%)
Control	3	0.5772±0.0321	-	DG3	3	0.4113±0.0536^c	32.268	DD1	3	0.3224±0.0113	–7.076
Model	3	0.3429±0.0313^a	-	DZ1	3	0.4356±0.0213^c	42.393	DD2	3	0.3542±0.0024	5.934
DG1	3	0.3778±0.0144	19.392	DZ2	3	0.4242±0.0334^c	34.104	DD3	3	0.3755±0.0157	19.323
DG2	3	0.3851±0.0329^b	20.478	DZ3	3	0.3266±0.0278	–1.977

Figure 1 oxidative stress-induced injured endothelial cells under different interventions (×200) Figures were photographed under an inverted microscope. A: conventional culture, no intervention; B: without drug intervention after modelling (1000μmol/L final concentration of H2O2-caused oxidative stress-damage); C: after modelling, treatment with Dangua Fang drug-containing serum; D: medium containing final concentrations of vitamin C of 20 μg/mL and coenzyme Q of 80 μg/mL. cells were cultured for 48 h.

Figure 2 Comparison of cell viability, apoptosis and cell cycle of oxidative stress-induced injured cell (n = 6,$\bar{x} \pm s$) A: comparison of cell viability; B: comparison of apoptosis; C: comparison of G2 + S. Control: conventional culture, no intervention; Model: without drug intervention after modelling (1000μmol/L final concentration of H2O2-caused oxidative stress-damage); Dangua: after modelling, treatment with Dangua Fang drug-containing serum; CoQ + Vit C: medium containing final concentrations of vitamin C of 20 μg/mL and coenzyme Q of 80 μg/mL. Cells were cultured for 48 h. compared with Control group: aP < 0.05; compared with Model group: bP < 0.01, cP < 0.05. G2 + S: Late stage of DNA synthesis + DNA synthesis stage.

Table 2 Influence of Dangua Fang on mitochondrial respiratory chain enzymes (μg/μL, $\bar{x} \pm s$)

Group	n	SDH	COX
Control	3	9.73±1.13	1.11±0.07
Model	3	11.91±0.77^a	0.78±0.04^a
Dangua	3	12.63±0.30	0.90±0.07^c
CoQ+VitC	3	6.92±0.18^b	0.82±0.04

Table 3 Comparison of Mitochondrial Membrane Potential Parameters in all groups ($\bar{x} \pm s$)

Group	n	R3 Red/green (×100)	R2 Red/green (×100)	R1 Red/green (×100)	Green (R2 + R3)
Contol	4	13.502±0.579	58.104±0.843	45.366±3.911	695.294±11.408
Model	4	3.658±0.012^a	47.802±0.502^a	6.513±0.130^a	887.108±3.724^a
Dangua	4	6.836±0.014^b	60.835±0.199^b	51.015±4.365^b	638.714±25.206^b
CoQ+VitC	4	10.129±0.009^b	73.697±1.041^b	61.643±0.636^b	686.668±14.191^b

Figure 3 Comparison of Na+-k+-ATPase and PGC-1αin all groups (n = 3, $\bar{x} \pm s$) A: comparison of Na+-k+-ATPase: Control group: conventional culture, no intervention; Model group: without drug intervention after modelling (1000μmol/L final concentration of H2O2-caused oxidative stress-damage); Dangua group: after modelling, treatment with Dangua Fang drug-containing serum; Diazoxide group: the culture solution contained a final diazoxide concentration of 0.1 μmol/mL; Combined: diazoxide + Dangua Fang. B: Comparsion of PGC-1α. The treatments of the control group, model group and Dangua group were the same as in Figure 3A. CoQ + Vit C group: medium containing final concentrations of vitamin C of 20 μg/mL and coenzyme Q of 80 μg/mL. Cells were cultured for 48 h. Compared with Control group, aP < 0.01, cP < 0.05; compared with Model group, bP < 0.01. PGC-1α: peroxisome proliferators-activated receptor-γcoactivator-1.

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