Cyanidin-3-O-glucoside plays a protective role against renal ischemia/ reperfusion injury via the JAK/STAT pathway

Renal dysfunction and tissue structural damage were significantly higher in the I/R group. After treatment with different concentrations of C3G, the levels of renal dysfunction and tissue structural damage decreased at different levels. And its protective effect was most pronounced at 200 mg/kg. The use of C3G reduced apoptosis as well as the expression of endoplasmic reticulum stress (ERS)-related proteins. Hypoxia/reoxygenation (H/R)-induced apoptosis and ERS are dependent on oxidative stress in vitro. In addition, both AG490 and C3G inhibited the activation of JAK/STAT pathway and attenuated oxidative stress, ischemia-induced apoptosis and ERS.

Conclusions:

The results demonstrated that C3G blocked renal apoptosis and ERS protein expression by preventing reactive oxygen species (ROS) production after I/R via the JAK/STAT pathway, suggesting that C3G may be a potential therapeutic agent for renal I/R injury.

Key words
Oxidative Stress; Apoptosis; Ischemia; Reperfusion; Apoptosis, Endoplasmic Reticulum Stress

Authorship

Yufeng Xiong

Conception and design the study

Technical procedures

Design of the study

Interpretation of data

Manuscript preparation

Renmin Hospital of Wuhan University – Department of Urology – Wuhan (Hubei), China.Renmin Hospital of Wuhan UniversityChinaWuhan, Hubei, ChinaRenmin Hospital of Wuhan University – Department of Urology – Wuhan (Hubei), China.

Renmin Hospital of Wuhan University – Institute of Urologic Disease – Wuhan (Hubei), China.Renmin Hospital of Wuhan UniversityChinaWuhan, Hubei, ChinaRenmin Hospital of Wuhan University – Institute of Urologic Disease – Wuhan (Hubei), China.

http://orcid.org/0000-0001-9810-1691

Jun Jian

Conception and design the study

Technical procedures

Design of the study

Interpretation of data

Statistical analysis

Critical revision

http://orcid.org/0000-0002-1406-4668

Honglin Yu

Conception and design the study

Technical procedures

Design of the study

Interpretation of data

Statistical analysis

University of Science and Technology of China, The First Affiliated Hospital – Department of Radiology – Hefei (Anhui), China.University of Science and Technology of ChinaChinaHefei, Anhui, ChinaUniversity of Science and Technology of China, The First Affiliated Hospital – Department of Radiology – Hefei (Anhui), China.

http://orcid.org/0000-0001-5330-2182

Jiejun Wu

Technical procedures

http://orcid.org/0009-0008-6094-1423

Hu Mao

Technical procedures

http://orcid.org/0000-0003-2252-5572

Ruikang Feng

Technical procedures

http://orcid.org/0000-0002-3228-3434

Lei Wang

Technical procedures

http://orcid.org/0000-0001-8412-1130

Yonghong Jian

Renmin Hospital of Wuhan University – Department of Nephrology – Wuhan (Hubei), China.Renmin Hospital of Wuhan UniversityChinaWuhan, Hubei, ChinaRenmin Hospital of Wuhan University – Department of Nephrology – Wuhan (Hubei), China.

http://orcid.org/0000-0002-9719-2103

Xiuheng Liu ^**Corresponding author: drliuxh@hotmail.com

Critical revision

http://orcid.org/0000-0003-3882-2715

*Corresponding author: drliuxh@hotmail.com

Conflict of interest

The authors declare no competing interests.

About the Authors

Yufeng Xiong is a Master in Medicine.

Jun Jian is a Master in Medicine.

Honglin Yu is a Medical Doctor.

Jiejun Wu is a Master in Medicine.

Hu Mao is a Medical Doctor.

Ruikang Feng is a Medical Doctor.

Lei Wang is a Medical Doctor.

Yonghong Jian is a Medical Doctor.

Xiuheng Liu is PhD.

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (6)

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Figure 1
C3G attenuated renal I/R injury. (a, b) Protective effects of C3G at doses of 50, 100, and 200 mg/kg on renal function in mice exposed to renal IR (n = 5 each); (c, d) Protective effect of C3G at different doses of 50, 100, and 200 mg/kg on renal tissue injury detected by H&E (×400) and randomly selected images from eight independent kidney samples used to quantify renal tubular injury scores. Values are expressed as medians (IQR) on the box plot. P < 0.05 indicates that the differences are statistically significant.

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Figure 2
C3G reduced apoptosis and ERS in vivo. (a) Representative images of TUNEL staining on kidney sections (×400); (b) Protein blot of Bax, Bcl-2 at 24 h of reperfusion; (c) and bar graph showed the fold change of Bax, Bcl-2 relative to the SHAM group in three independent samples; (d) Protein blots of GRP78, CHOP at 24 h of reperfusion; (e) Bar graph showed the change in GRP78, CHOP ploidy. Values are expressed as medians (IQR) on the box plot. P < 0.05 indicates that the differences are statistically significant.

Thumbnail

Figure 3
H/R-induced apoptosis and ERS depend on oxidative stress in vitro. HK-2 cells were pretreated with 5 mmol/L NAC for 1 h followed by H/R. (a) ROS production observed by fluorescence microscopy (×400); (b) Protein blot analysis of Bax, BCL-2 expression; (c) Bax, BCL-2 protein expression was quantified by optical densitometry and normalized to the expression of GAPDH from three independent experiments; (d) Protein blot analysis of GRP78, CHOP expression; (e) GRP78, CHOP protein expression was quantified by optical densitometry normalized to the expression of GAPDH from three independent experiments. Values are expressed as medians (IQR) on the box plot. p < 0.05 indicates that the differences are statistically significant.

Thumbnail

Figure 4
C3G attenuated H/R-induced oxidative stress, apoptosis and ERS in vitro. (a) HK-2 cells were treated with different concentrations (50, 100, 150, 200 µmol/L) of C3G for 24h, and then their cell viability was detected by CCK-8; (b) HK-2 cells were treated with different concentrations (50, 100, 150, 200 µmol/L) of C3G for 24 h to establish the H/R model, and then their cell viability was detected by CCK-8; (c) ROS production observed by fluorescence microscopy (×400); (d) Apoptosis was assessed by flow cytometry; (e) Protein blot analysis of GRP78, CHOP expression; (f) GRP78, CHOP protein expression was quantified by optical densitometry normalized to the expression of GAPDH from three independent experiments. Values are expressed as medians (IQR) on the box plot. P < 0.05 indicates that the differences are statistically significant. ns, no significance.

Thumbnail

Figure 5
C3G attenuated renal injury by inhibiting the JAK/STAT pathway. (a, c) Protein blot analysis of protein expression of JAK2, p-JAK2, STAT3, p-STAT3; and (b, d) quantitative analysis of p-JAK2 and p-STAT3; (e, f) MDA, SOD production in mice. Values are expressed as medians (IQR) on the box plot. P < 0.05 indicates that the differences are statistically significant.

Thumbnail

Figure 6
C3G attenuated renal injury by inhibiting the JAK/STAT pathway. (a, b) Protein expression of JAK2, p-JAK2, STAT3, p-STAT3 in cells of different treatment groups; (c, d) Quantitative analysis of p-JAK2 and p-STAT3; (e) Protein blot analysis of GRP78 and CHOP protein expression in the indicated groups; (f) Quantitative analysis of GRP78 and CHOP; (g) Representative protein blot analysis of Bax, Bcl-2 in the indicated groups. Quantitative analysis of Bax and Bcl-2.Values are expressed as medians (IQR) on the box plot. P < 0.05 indicates that the differences are statistically significant.

Figure 1 C3G attenuated renal I/R injury. (a, b) Protective effects of C3G at doses of 50, 100, and 200 mg/kg on renal function in mice exposed to renal IR (n = 5 each); (c, d) Protective effect of C3G at different doses of 50, 100, and 200 mg/kg on renal tissue injury detected by H&E (×400) and randomly selected images from eight independent kidney samples used to quantify renal tubular injury scores. Values are expressed as medians (IQR) on the box plot. P < 0.05 indicates that the differences are statistically significant.

Figure 2 C3G reduced apoptosis and ERS in vivo. (a) Representative images of TUNEL staining on kidney sections (×400); (b) Protein blot of Bax, Bcl-2 at 24 h of reperfusion; (c) and bar graph showed the fold change of Bax, Bcl-2 relative to the SHAM group in three independent samples; (d) Protein blots of GRP78, CHOP at 24 h of reperfusion; (e) Bar graph showed the change in GRP78, CHOP ploidy. Values are expressed as medians (IQR) on the box plot. P < 0.05 indicates that the differences are statistically significant.

Figure 3 H/R-induced apoptosis and ERS depend on oxidative stress in vitro. HK-2 cells were pretreated with 5 mmol/L NAC for 1 h followed by H/R. (a) ROS production observed by fluorescence microscopy (×400); (b) Protein blot analysis of Bax, BCL-2 expression; (c) Bax, BCL-2 protein expression was quantified by optical densitometry and normalized to the expression of GAPDH from three independent experiments; (d) Protein blot analysis of GRP78, CHOP expression; (e) GRP78, CHOP protein expression was quantified by optical densitometry normalized to the expression of GAPDH from three independent experiments. Values are expressed as medians (IQR) on the box plot. p < 0.05 indicates that the differences are statistically significant.

Figure 4 C3G attenuated H/R-induced oxidative stress, apoptosis and ERS in vitro. (a) HK-2 cells were treated with different concentrations (50, 100, 150, 200 µmol/L) of C3G for 24h, and then their cell viability was detected by CCK-8; (b) HK-2 cells were treated with different concentrations (50, 100, 150, 200 µmol/L) of C3G for 24 h to establish the H/R model, and then their cell viability was detected by CCK-8; (c) ROS production observed by fluorescence microscopy (×400); (d) Apoptosis was assessed by flow cytometry; (e) Protein blot analysis of GRP78, CHOP expression; (f) GRP78, CHOP protein expression was quantified by optical densitometry normalized to the expression of GAPDH from three independent experiments. Values are expressed as medians (IQR) on the box plot. P < 0.05 indicates that the differences are statistically significant. ns, no significance.

Figure 5 C3G attenuated renal injury by inhibiting the JAK/STAT pathway. (a, c) Protein blot analysis of protein expression of JAK2, p-JAK2, STAT3, p-STAT3; and (b, d) quantitative analysis of p-JAK2 and p-STAT3; (e, f) MDA, SOD production in mice. Values are expressed as medians (IQR) on the box plot. P < 0.05 indicates that the differences are statistically significant.

Figure 6 C3G attenuated renal injury by inhibiting the JAK/STAT pathway. (a, b) Protein expression of JAK2, p-JAK2, STAT3, p-STAT3 in cells of different treatment groups; (c, d) Quantitative analysis of p-JAK2 and p-STAT3; (e) Protein blot analysis of GRP78 and CHOP protein expression in the indicated groups; (f) Quantitative analysis of GRP78 and CHOP; (g) Representative protein blot analysis of Bax, Bcl-2 in the indicated groups. Quantitative analysis of Bax and Bcl-2.Values are expressed as medians (IQR) on the box plot. P < 0.05 indicates that the differences are statistically significant.

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Cyanidin-3-O-glucoside plays a protective role against renal ischemia/ reperfusion injury via the JAK/STAT pathway

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[1] *Corresponding author: drliuxh@hotmail.com