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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (1): 85-90.doi: 10.3736/jcim20120113

• Original Experimental Research • Previous Articles     Next Articles

Chinese herbal medicine Lingqi Huangban Granule protects retinal pigment epithelial cells against oxidative stress-induced injury in vitro

Li Cai-hong1,Qiu Qing-hua2(),Wu Xing-wei2,Gong Yuan-yuan2,Xie Zheng-gao3,Song Yi4,Gu Qing2   

  1. 1. Department of Ophthalmology, Zhengzhou Central Hospital, Zhengzhou 450007, Henan Province, China
    2. Department of Ophthalmology, Shanghai First People’s Hospital, Shanghai Jiao Tong University, Shanghai 200080, China;
    3. Department of Ophthalmology, Northern Jiangsu People’s Hospital, Yangzhou 225001, Jiangsu Province, China;
    4. Department of Ophthalmology, Shanghai Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200072, China
  • Received:2011-06-28 Accepted:2011-09-20 Online:2012-01-20 Published:2018-09-21
  • Contact: Qiu Qing-hua

OBJECTIVE: To observe the protective effects of drug-contained serum of Lingqi Huangban Granule (LQHBG), a compound traditional Chinese herbal medicine, on oxidative stress-induced injury in rabbit retinal pigment epithelial (RPE) cells in vitro.METHODS: The oxidative stress of rabbit RPE cells in vitro was induced with hydrogen peroxide (500 μmol/L) and different concentrations of LQHBG were administered to rats to prepare medicated serum. RPE cells were randomized into normal control group (no hydrogen peroxide), model group (hydrogen peroxide), model plus serum group (hydrogen peroxide and 10% control serum), model plus low-dose LQHBG group (hydrogen peroxide and low-dose LQHBG-medicated serum) and model plus high-dose LQHBG group (hydrogen peroxide and high-dose LQHBG-medicated serum). Teminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and flow cytometry (FCM) were used to measure apoptosis of cultured rabbit RPE cells. Protein expressions of caspase-3 and Bcl-XL were observed by Western blot method.RESULTS: FCM results showed that the apoptotic rates of the normal control group, model group, control serum group and serum containing low- and high-dose LQHBG groups were (4.85±0.26) %, (20.02±1.37) %, (21.84±0.94) %, (13.56±0.55) %, and (8.58±0.39) %, respectively; compared with the model group, the apoptotic rates of RPE cells in the low- and high-dose LQHBG groups were obviously reduced in a dose-related manner (P<0.05). TUNEL results showed that nuclei of apoptotic cells were stained brown; the number of apoptotic cells in the low- and high-dose LQHBG groups was obviously less than that in the model group. The protein expression of caspase-3 was up-regulated in the model and control serum groups, which was higher than that in the high-dose LQHBG group (P<0.05). The protein expression of Bcl-XL was down-regulated in the model and control serum groups, which was lower than that in the low- and high-dose LQHBG groups (P<0.01).CONCLUSION: Drug-contained serum of LQHBG obviously reduces apoptosis and partly protects rabbit RPE cells from oxidative stress-induced injury. The protective function is due to an improvement in antioxidant abilities, down-regulation of the expression of caspase-3 and up-regulation of the expression of Bcl-XL.

Key words: drugs, Chinese herbal, age-related macular degeneration, retinal pigment epithelial cell, oxidative stress, serologic pharmacology, apoptosis, in vitro

Figure 1

Apoptosis of rabbit retinal pigment epithelial cells in each group measured by TUNEL assay (Confocal microscopy, ×400) No TUNEL-positive cells were noticed in the normal control group (A); there were abundant yellow fluorescent nuclei in the model group (B) and the 10% control serum group (C); sparse yellow fluorescent nuclei were found in the low-dose serum containing Lingqi Huangban group (D); few yellow fluorescent nuclei were found in the high-dose serum containing Lingqi Huangban group (E). TUNEL: teminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling."

Table 1

Relative expressions of caspase-3 and Bcl-XL proteins in each group detected by Western blot method"

Group n Caspase-3 protein/β-actin Bcl-XL protein/β-actin
Normal control 3 0.20±0.03 0.49±0.06
Model 3 0.47±0.05** 0.24±0.03**
10% Control serum 3 0.62±0.06**△ 0.38±0.04*△
Low-dose serum containing Lingqi Huangban 3 0.44±0.04 0.52±0.03△△▲
High-dose serum containing Lingqi Huangban 3 0.36±0.04△▲▲□ 0.75±0.08△△▲▲□□

Figure 2

Expressions of caspase-3 and Bcl-XL proteins in each group detected by Western blot methodEqual protein loading was confirmed with the β-actin antibody. A: Normal control group; B: Model group; C: 10% control serum group; D: Low-dose serum containing Lingqi Huangban group; E: High-dose serum containing Lingqi Huangban group."

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