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International Journal of Genomics Volume 2017 ,2017-11-29
HuoXueJieDu Formula Alleviates Diabetic Retinopathy in Rats by Inhibiting SOCS3-STAT3 and TIMP1-A2M Pathways
Research Article
Hongliang Wang 1 , 2 Wei Xing 1 Shijie Tang 3 Zhenglin Wang 1 Tiantian Lv 1 Yan Wu 4 Shuzhen Guo 1 Chun Li 5 Jing Han 4 Ruixin Zhu 6 Wei Wang 1
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DOI:10.1155/2017/4832125
Received 2017-03-30, accepted for publication 2017-10-08, Published 2017-10-08
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摘要

HuoXueJieDu (HXJD) formula exerts protective effects against diabetic retinopathy (DR) in rats, but its underlying mechanism remains unknown. In the present study, the diabetic rats were established using streptozocin. The administration of HXJD was initiated at 20 weeks after diabetes induction and continued for 12 weeks. Whole genome expression profiles in rat retinas were examined using microarray technology. Differential gene expression and pathway enrichment analysis were conducted on the microarray data, with validation through real-time PCR and immunohistochemical staining. The results showed that 170 genes and several IPA canonical pathways related to inflammation, matrix metabolism, and phototransduction were regulated by HXJD. PCR validation of selected genes, including SOCS3, STAT3, TIMP1, and A2M, confirmed the gene expression changes influenced by HXJD. In addition, the immunohistochemical staining results suggested that critical members of the SOCS3-STAT3 pathway were also affected by HXJD. Taken together, these results indicated that SOCS3-STAT3 and TIMP1-A2M pathways might mediate the alleviation of HXJD activities in rats with diabetic retinopathy.

授权许可

Copyright © 2017 Hongliang Wang et al. 2017
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

图表

The effect of HXJD on retinal morphology. The retinas were stained with haematoxylin and eosin staining. (a) Retinas of normal rats. Retinal ganglion cell (RGC), inner plexiform layer (IPL), and photoreceptor layer (PL). (b) Retinas of diabetic rats. (c) Retinas of the rats in the HXJD group. Magnification: ×400. (d) The retinal thickness from IPL to PL in the midretina was measured. HXJD increased the retinal thickness. (e) The RGC number was counted. Retinas of the rats in the HXJD group showed an increased number of RGC. Data are represented as the means ± SEM. (n=3~5). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001 compared with the diabetic group.

The effect of HXJD on retinal morphology. The retinas were stained with haematoxylin and eosin staining. (a) Retinas of normal rats. Retinal ganglion cell (RGC), inner plexiform layer (IPL), and photoreceptor layer (PL). (b) Retinas of diabetic rats. (c) Retinas of the rats in the HXJD group. Magnification: ×400. (d) The retinal thickness from IPL to PL in the midretina was measured. HXJD increased the retinal thickness. (e) The RGC number was counted. Retinas of the rats in the HXJD group showed an increased number of RGC. Data are represented as the means ± SEM. (n=3~5). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001 compared with the diabetic group.

The effect of HXJD on retinal morphology. The retinas were stained with haematoxylin and eosin staining. (a) Retinas of normal rats. Retinal ganglion cell (RGC), inner plexiform layer (IPL), and photoreceptor layer (PL). (b) Retinas of diabetic rats. (c) Retinas of the rats in the HXJD group. Magnification: ×400. (d) The retinal thickness from IPL to PL in the midretina was measured. HXJD increased the retinal thickness. (e) The RGC number was counted. Retinas of the rats in the HXJD group showed an increased number of RGC. Data are represented as the means ± SEM. (n=3~5). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001 compared with the diabetic group.

The effect of HXJD on retinal morphology. The retinas were stained with haematoxylin and eosin staining. (a) Retinas of normal rats. Retinal ganglion cell (RGC), inner plexiform layer (IPL), and photoreceptor layer (PL). (b) Retinas of diabetic rats. (c) Retinas of the rats in the HXJD group. Magnification: ×400. (d) The retinal thickness from IPL to PL in the midretina was measured. HXJD increased the retinal thickness. (e) The RGC number was counted. Retinas of the rats in the HXJD group showed an increased number of RGC. Data are represented as the means ± SEM. (n=3~5). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001 compared with the diabetic group.

The effect of HXJD on retinal morphology. The retinas were stained with haematoxylin and eosin staining. (a) Retinas of normal rats. Retinal ganglion cell (RGC), inner plexiform layer (IPL), and photoreceptor layer (PL). (b) Retinas of diabetic rats. (c) Retinas of the rats in the HXJD group. Magnification: ×400. (d) The retinal thickness from IPL to PL in the midretina was measured. HXJD increased the retinal thickness. (e) The RGC number was counted. Retinas of the rats in the HXJD group showed an increased number of RGC. Data are represented as the means ± SEM. (n=3~5). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001 compared with the diabetic group.

The effect of HXJD on the expression of genes associated with cytokine signalling and matrix metalloprotease expression. (a) Heat map of the regulated genes in normal, diabetic, and diabetic +HXJD rat groups. (b–g) The microarray data were validated using real-time PCR. Data are represented as the means ± SEM. (n=3~4). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001, compared with the diabetic group.

The effect of HXJD on the expression of genes associated with cytokine signalling and matrix metalloprotease expression. (a) Heat map of the regulated genes in normal, diabetic, and diabetic +HXJD rat groups. (b–g) The microarray data were validated using real-time PCR. Data are represented as the means ± SEM. (n=3~4). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001, compared with the diabetic group.

The effect of HXJD on the expression of genes associated with cytokine signalling and matrix metalloprotease expression. (a) Heat map of the regulated genes in normal, diabetic, and diabetic +HXJD rat groups. (b–g) The microarray data were validated using real-time PCR. Data are represented as the means ± SEM. (n=3~4). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001, compared with the diabetic group.

The effect of HXJD on the expression of genes associated with cytokine signalling and matrix metalloprotease expression. (a) Heat map of the regulated genes in normal, diabetic, and diabetic +HXJD rat groups. (b–g) The microarray data were validated using real-time PCR. Data are represented as the means ± SEM. (n=3~4). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001, compared with the diabetic group.

The effect of HXJD on the expression of genes associated with cytokine signalling and matrix metalloprotease expression. (a) Heat map of the regulated genes in normal, diabetic, and diabetic +HXJD rat groups. (b–g) The microarray data were validated using real-time PCR. Data are represented as the means ± SEM. (n=3~4). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001, compared with the diabetic group.

The effect of HXJD on the expression of genes associated with cytokine signalling and matrix metalloprotease expression. (a) Heat map of the regulated genes in normal, diabetic, and diabetic +HXJD rat groups. (b–g) The microarray data were validated using real-time PCR. Data are represented as the means ± SEM. (n=3~4). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001, compared with the diabetic group.

The effect of HXJD on the expression of genes associated with cytokine signalling and matrix metalloprotease expression. (a) Heat map of the regulated genes in normal, diabetic, and diabetic +HXJD rat groups. (b–g) The microarray data were validated using real-time PCR. Data are represented as the means ± SEM. (n=3~4). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001, compared with the diabetic group.

HXJD reduces the protein expression of p-STAT3, STAT3, SOCS3, A2M, and TIMP1 in the retinas of diabetic rats. The protein expression of p-STAT3, STAT3, SOCS3, A2M, and TIMP1 were measured using western blot. (a) Western blot images. (b–f) Image analysis of western blot. Data are represented as the means ± SEM. (n=3). ∗P<0.05 and ∗∗∗P<0.001, compared with the diabetic group.

HXJD reduces the protein expression of p-STAT3, STAT3, SOCS3, A2M, and TIMP1 in the retinas of diabetic rats. The protein expression of p-STAT3, STAT3, SOCS3, A2M, and TIMP1 were measured using western blot. (a) Western blot images. (b–f) Image analysis of western blot. Data are represented as the means ± SEM. (n=3). ∗P<0.05 and ∗∗∗P<0.001, compared with the diabetic group.

HXJD reduces the protein expression of p-STAT3, STAT3, SOCS3, A2M, and TIMP1 in the retinas of diabetic rats. The protein expression of p-STAT3, STAT3, SOCS3, A2M, and TIMP1 were measured using western blot. (a) Western blot images. (b–f) Image analysis of western blot. Data are represented as the means ± SEM. (n=3). ∗P<0.05 and ∗∗∗P<0.001, compared with the diabetic group.

HXJD reduces the protein expression of p-STAT3, STAT3, SOCS3, A2M, and TIMP1 in the retinas of diabetic rats. The protein expression of p-STAT3, STAT3, SOCS3, A2M, and TIMP1 were measured using western blot. (a) Western blot images. (b–f) Image analysis of western blot. Data are represented as the means ± SEM. (n=3). ∗P<0.05 and ∗∗∗P<0.001, compared with the diabetic group.

HXJD reduces the protein expression of p-STAT3, STAT3, SOCS3, A2M, and TIMP1 in the retinas of diabetic rats. The protein expression of p-STAT3, STAT3, SOCS3, A2M, and TIMP1 were measured using western blot. (a) Western blot images. (b–f) Image analysis of western blot. Data are represented as the means ± SEM. (n=3). ∗P<0.05 and ∗∗∗P<0.001, compared with the diabetic group.

HXJD reduces the protein expression of p-STAT3, STAT3, SOCS3, A2M, and TIMP1 in the retinas of diabetic rats. The protein expression of p-STAT3, STAT3, SOCS3, A2M, and TIMP1 were measured using western blot. (a) Western blot images. (b–f) Image analysis of western blot. Data are represented as the means ± SEM. (n=3). ∗P<0.05 and ∗∗∗P<0.001, compared with the diabetic group.

HXJD reduces the expression of JAK2, p38 MAPK, and NF-κB in the retinas of diabetic rats. Distribution and expression of JAK2, p38 MAPK, and NF-κB were measured using IHC. (a–c) IHC staining of JAK2. (d–f) IHC staining of p38 MAPK. (g–i) IHC staining of NF-κB. Magnification: ×400. (j–l) Image analysis of JAK2, p38 MAPK, and NF-κB. A reduction of JAK2, p38 MAPK, and NF-κB was observed in the HXJD group. Data are represented as the means ± SEM. (n=3~6). ∗P<0.05 and ∗∗P<0.01, compared with the diabetic group.

The molecular mechanism of HDJX in attenuating diabetic retinopathy in rats via the inhibition of SOCS3-STAT3 and T1MP1-A2M pathways. JAK2, NF-κB, and p38 MAPK promote SOCS3-STAT3 signalling, then SOCS3-STAT3 is involved in the progression of DR. TIMP1 and A2M play roles in matrix degradation which also take part in the process of DR. HXJD has the ability of inhibiting these pathological changes and thereafter it will ameliorate DR.

通讯作者

1. Jing Han.Institute of Chinese Medicine, Beijing University of Chinese medicine, Beijing 100029, China, bucm.edu.cn.hanjing8585@163.com
2. Ruixin Zhu.Department of Bioinformatics, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China, tongji.edu.cn.rxzhu@tongji.edu.cn
3. Wei Wang.College of Basic Medicine, Key Laboratory of Ministry of Education (Syndromes and Formulas), Key Laboratory of Beijing (Syndromes and Formulas), Beijing University of Chinese medicine, Beijing 100029, China, bucm.edu.cn.wangwei26960@126.com

推荐引用方式

Hongliang Wang,Wei Xing,Shijie Tang,Zhenglin Wang,Tiantian Lv,Yan Wu,Shuzhen Guo,Chun Li,Jing Han,Ruixin Zhu,Wei Wang. HuoXueJieDu Formula Alleviates Diabetic Retinopathy in Rats by Inhibiting SOCS3-STAT3 and TIMP1-A2M Pathways. International Journal of Genomics ,Vol.2017(2017)

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