艾灸对DM性ED大鼠阴茎勃起功能信号通路影响研究
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摘要
目的：
观察艾灸对糖尿病(DM)性阴茎勃起功能障碍(ED)大鼠阴茎勃起功能、血糖、一氧化氮合酶(NOS)、环磷酸鸟苷(cGMP)、RhoA/Rho激酶(ROCKII)的影响,探讨艾灸对DM-ED阴茎勃起功能及NO-cGMP、RhoA/Rho激酶信号通路的影响,为DM-ED的艾灸治疗提供实验及现代理论依据。方法：
以链脲佐菌素(STZ)诱导建立DM大鼠模型；DM动物模型成功者,以阿朴吗啡(APO)阴茎勃起实验,筛选DM性ED大鼠模型。以正常大鼠为空白组；发生ED者随机分为模型组、模灸组。模灸组给予“肾俞”、“足三里”麦粒灸治疗。以APO检测治疗前后大鼠阴茎勃起变化；采用罗康全血糖仪检测对大鼠血糖的影响；采用硝酸还原酶法检测大鼠阴茎NOS；采用放免法检测大鼠阴茎cGMP;采用RT-PCR、免疫组化、Western bloting检测对大鼠阴茎RhoA、Rho激酶(ROCKII)表达的影响。结果：
1)阴茎勃起治疗后模灸组与模型组相比,勃起动物数、勃起率改善(P<0.05),总勃起次数改善(P>0.05)；模灸组、模型组勃起动物数、总勃起次数、勃起率均差于空白组(P<0.01)。
2)血糖治疗前模灸组与模型组相比无显著差异(P＞0.05)。治疗后模灸组血糖水平低于模型组(P<0.05)；空白组血糖远低于模灸组、模型组(P<0.01)。
3)NOS治疗后模灸组NOS高于模型组(总NOS、cNOS P＜0.01, iNOS P<0.05),低于空白组(总NOS P＜0.05, iNOS、cNOS P＞0.05);模型组NOS均低于空白组(P 4) cGMP模型组cGMP水平低于空白组,比较有显著性差异(P<0.01)；治疗后模灸组cGMP水平高于模型组(P<0.01),与空白组相比没有显著差异(P>0.05)。
5) RhoA RT-PCR:治疗后模型组与空白组比较,阴茎RhoA mRNA表达明显升高(P<0.05),模灸组与模型组比较,阴茎RhoA mRNA表达低于模型组,但无显著性差异(P>0.05)。模灸组与空白组比较无显著性差异(P>0.05)。
免疫组化：治疗后模型组、模灸组与空白组比较,阴茎RhoA蛋白阳性表达明显升高(P<0.01),模灸组阴茎RhoA蛋白阳性表达明显弱于模型组(P<0.01)。
Western blot:治疗后模型组、模灸组与空白组比较,阴茎RhoA蛋白表达明显升高(P<0.01)；模灸组与模型组比较,阴茎RhoA蛋白表达低于模型组(P<0.01)。
6)Rho激酶(ROCKII) RT-PCR:治疗后模型组、模灸组与空白组比较,阴茎ROCKII mRNA表达明显升高(模型组P<0.01、模灸组P<0.05),模灸组阴茎ROCKII mRNA表达低于模型组,但无显著性差异(P>0.05)。
免疫组化：治疗后模型组、模灸组与空白组比较,阴茎ROCKII蛋白阳性表达明显升高(P<0.01)；模灸组阴茎ROCKII蛋白阳性表达明显弱于模型组(P<0.05)。
Western blot:治疗后模型组、模灸组与空白组比较,阴茎ROCKII蛋白表达明显升高(模型组P<0.01、模灸组P<0.05)；模灸组阴茎ROCKII蛋白表达低于模型组(P<0.05)。
结论：
1)艾灸能改善DM性ED大鼠血糖水平及其阴茎勃起功能障碍。
2)艾灸能提高DM性ED大鼠阴茎组织总NOS、cNOS、iNOS、cGMP含量,艾灸提高NO-cGMP水平可能是艾灸改善DM性ED大鼠阴茎勃起功能障碍作用机制之一。
3)艾灸能降低DM性ED大鼠阴茎组织RhoA、Rho激酶水平,艾灸降低RhoA/Rho激酶水平可能是艾灸改善DM性ED大鼠阴茎勃起功能障碍作用机制之一
Objective:To investigate the role of the NO-cGMP and RhoA/Rho signal pathway in the erectile function of rats with diabetic ED treated with moxibustion and provide experimental and theoretical evidence for clinical treatment by studying the influence of moxibustion on the erectile function,the level of blood glucose,the activity of NOS,the concentration of cGMP and the expression of RhoA/Rho kinase(ROCKII) in the penis of rats with diabetic ED.
Methods:Male Sprague-Dawley rats with normal erectile function were injected with streptozotozin(STZ) to make experimental models of Diabetes Mellitus(DM).Then apomorphine(APO) was injected into all of the DM rats to select ED rats models with DM. The normal rats were in the blank group and the ED rats were randomly divided into the model group and the moxibustion group. The model group were not being treated and the model-moxibustion group were treated with moxibustion on acupoint "Shenshu" (BL23) and "Zusanli"(ST36). The erection induced by APO was observed before and after the treatment. The blood glucose was examined before and after the treatment. The activity of NOS in the penis was measured with the nitrate reductase method(spectrophotometric analysis).The concentration of cGMP in the penis was measured with radioimmunoassay. The expression of RhoA and Rho kinase (ROCKII) in the penis was measured with RT-PCR, immunohistochemistry and Western blot technique, respectively.
Results:
1.The counts of rats which had the ability of erection (P＜0.05), the erection rate (P＜0.05) and the total frequency of erection (P＞0.05) in the model-moxibustion group were superior to the model group after the treatment. And the counts of rats which had the ability of erection, the erection rate and the total frequency of erection in the model-moxibustion and the model group were inferior to the blank group (P＜0.01).
2.There was no difference in the level of blood glucose between the model-moxibustion group and the model group (P＞0.05) before the treatment. The level of blood glucose in the model-moxibustion was lower than the model group (P＜0.05) and The level of blood glucose in the blank group was much lower than that in the model-moxibustion group and the model group (P＜0.01) after the treatment.
3.The activity of NOS in the penis in the model-moxibustion group was higher than that in the model group (total NOS,cNOS P＜0.01,iNOS P＜0.05) and lower than that in the blank group(total NOS P＜0.05,cNOS,cNOS P＞0.05) after the treatment. The activity of NOS in the model group was much lower than that in the blank group after the treatment (P＜0.01).
4.After the treatment, there was no difference in the concentration of cGMP in the penis between the model-moxibustion group and the blank group (P＞0.05) and the concentration of cGMP in the two groups were much higher than that in the model group (P＜0.01).
5.After the treatment, the expression of RhoA mRNA in the penis in the model group measured with RT-PCR increased significantly, compared with that in the blank group (P＜0.05). The expression of RhoA mRNA in the model-moxibustion group was lower than that in the model group, though there was no difference between the two groups (P＞0.05). And there was no difference in the expression of RhoA mRNA between the model-moxibustion group and the blank group (P＞0.05)
The expression of RhoA protein in the penis measured with immunohistochemistry technique increased significantly in the model-moxibustion group and the model group, compared with that in the blank group (P＜0.01) after the treatment. And the expression of RhoA protein in the model-moxibustion group was much lower than that in the model group (P＜0.01).
The expression of RhoA protein in the penis in the model-moxibustion group and the model group measured with Western blot increased significantly, compared with that in the blank group (P＜0.01) after the treatment. And the expression of RhoA protein in the model-moxibustion group was much lower than that in the model group (P<0.01).
6:After the treatment, the expression of ROCKII mRNA in the penis measured with RT-PCR increased significantly in the model-moxibustion group(P＜0.05) and the model group (P＜0.01), compared with that in the blank group. And the expression of ROCKII mRNA in the model-moxibustion group was lower than that in the model group,though there was no difference between the two groups (P＞0.05)
The expression of ROCKII protein in the penis tissue measured with immunohistochemistry technique increased significantly in the model-moxibustion group and the model group, compared with that in the blank group (P＜0.01) after the treatment. And the expression of ROCKII protein in the model-moxibustion group was lower than that in the model group (P＜0.05).
The expression of ROCKII protein in the penis measured with Western blot increased significantly in the model-moxibustion group(P＜0.05) and the model group (P＜0.01), compared with that in the blank group after the treatment. And the expression of ROCKII mRNA in the model-moxibustion group was lower than that in the model group (P＜0.05).
Conclusions:
1.Moxibustion could decrease the level of blood glucose and promote the erectile function of rats with diabetic ED.
2.Moxibustion could increase the activity of total NOS, cNOS and iNOS and the concentration of cGMP in the penis of rats with diabetic ED, which proved that the NO-cGMP signal pathway might play a role in improving the erectile function when the rats were treated with moxibustion.
3.Moxibustion could decrease the expression of RhoA/Rho kinase in the penis of rats with diabetic ED, which proved that the RhoA/Rho kinase signal pathway might also play a role in improving the erectile function when the rats were treated with moxibustion.
观察艾灸对糖尿病(DM)性阴茎勃起功能障碍(ED)大鼠阴茎勃起功能、血糖、一氧化氮合酶(NOS)、环磷酸鸟苷(cGMP)、RhoA/Rho激酶(ROCKII)的影响,探讨艾灸对DM-ED阴茎勃起功能及NO-cGMP、RhoA/Rho激酶信号通路的影响,为DM-ED的艾灸治疗提供实验及现代理论依据。方法：
以链脲佐菌素(STZ)诱导建立DM大鼠模型；DM动物模型成功者,以阿朴吗啡(APO)阴茎勃起实验,筛选DM性ED大鼠模型。以正常大鼠为空白组；发生ED者随机分为模型组、模灸组。模灸组给予“肾俞”、“足三里”麦粒灸治疗。以APO检测治疗前后大鼠阴茎勃起变化；采用罗康全血糖仪检测对大鼠血糖的影响；采用硝酸还原酶法检测大鼠阴茎NOS；采用放免法检测大鼠阴茎cGMP;采用RT-PCR、免疫组化、Western bloting检测对大鼠阴茎RhoA、Rho激酶(ROCKII)表达的影响。结果：
1)阴茎勃起治疗后模灸组与模型组相比,勃起动物数、勃起率改善(P<0.05),总勃起次数改善(P>0.05)；模灸组、模型组勃起动物数、总勃起次数、勃起率均差于空白组(P<0.01)。
2)血糖治疗前模灸组与模型组相比无显著差异(P＞0.05)。治疗后模灸组血糖水平低于模型组(P<0.05)；空白组血糖远低于模灸组、模型组(P<0.01)。
3)NOS治疗后模灸组NOS高于模型组(总NOS、cNOS P＜0.01, iNOS P<0.05),低于空白组(总NOS P＜0.05, iNOS、cNOS P＞0.05);模型组NOS均低于空白组(P
5) RhoA RT-PCR:治疗后模型组与空白组比较,阴茎RhoA mRNA表达明显升高(P<0.05),模灸组与模型组比较,阴茎RhoA mRNA表达低于模型组,但无显著性差异(P>0.05)。模灸组与空白组比较无显著性差异(P>0.05)。
免疫组化：治疗后模型组、模灸组与空白组比较,阴茎RhoA蛋白阳性表达明显升高(P<0.01),模灸组阴茎RhoA蛋白阳性表达明显弱于模型组(P<0.01)。
Western blot:治疗后模型组、模灸组与空白组比较,阴茎RhoA蛋白表达明显升高(P<0.01)；模灸组与模型组比较,阴茎RhoA蛋白表达低于模型组(P<0.01)。
6)Rho激酶(ROCKII) RT-PCR:治疗后模型组、模灸组与空白组比较,阴茎ROCKII mRNA表达明显升高(模型组P<0.01、模灸组P<0.05),模灸组阴茎ROCKII mRNA表达低于模型组,但无显著性差异(P>0.05)。
免疫组化：治疗后模型组、模灸组与空白组比较,阴茎ROCKII蛋白阳性表达明显升高(P<0.01)；模灸组阴茎ROCKII蛋白阳性表达明显弱于模型组(P<0.05)。
Western blot:治疗后模型组、模灸组与空白组比较,阴茎ROCKII蛋白表达明显升高(模型组P<0.01、模灸组P<0.05)；模灸组阴茎ROCKII蛋白表达低于模型组(P<0.05)。
结论：
1)艾灸能改善DM性ED大鼠血糖水平及其阴茎勃起功能障碍。
2)艾灸能提高DM性ED大鼠阴茎组织总NOS、cNOS、iNOS、cGMP含量,艾灸提高NO-cGMP水平可能是艾灸改善DM性ED大鼠阴茎勃起功能障碍作用机制之一。
3)艾灸能降低DM性ED大鼠阴茎组织RhoA、Rho激酶水平,艾灸降低RhoA/Rho激酶水平可能是艾灸改善DM性ED大鼠阴茎勃起功能障碍作用机制之一
Objective:To investigate the role of the NO-cGMP and RhoA/Rho signal pathway in the erectile function of rats with diabetic ED treated with moxibustion and provide experimental and theoretical evidence for clinical treatment by studying the influence of moxibustion on the erectile function,the level of blood glucose,the activity of NOS,the concentration of cGMP and the expression of RhoA/Rho kinase(ROCKII) in the penis of rats with diabetic ED.
Methods:Male Sprague-Dawley rats with normal erectile function were injected with streptozotozin(STZ) to make experimental models of Diabetes Mellitus(DM).Then apomorphine(APO) was injected into all of the DM rats to select ED rats models with DM. The normal rats were in the blank group and the ED rats were randomly divided into the model group and the moxibustion group. The model group were not being treated and the model-moxibustion group were treated with moxibustion on acupoint "Shenshu" (BL23) and "Zusanli"(ST36). The erection induced by APO was observed before and after the treatment. The blood glucose was examined before and after the treatment. The activity of NOS in the penis was measured with the nitrate reductase method(spectrophotometric analysis).The concentration of cGMP in the penis was measured with radioimmunoassay. The expression of RhoA and Rho kinase (ROCKII) in the penis was measured with RT-PCR, immunohistochemistry and Western blot technique, respectively.
Results:
1.The counts of rats which had the ability of erection (P＜0.05), the erection rate (P＜0.05) and the total frequency of erection (P＞0.05) in the model-moxibustion group were superior to the model group after the treatment. And the counts of rats which had the ability of erection, the erection rate and the total frequency of erection in the model-moxibustion and the model group were inferior to the blank group (P＜0.01).
2.There was no difference in the level of blood glucose between the model-moxibustion group and the model group (P＞0.05) before the treatment. The level of blood glucose in the model-moxibustion was lower than the model group (P＜0.05) and The level of blood glucose in the blank group was much lower than that in the model-moxibustion group and the model group (P＜0.01) after the treatment.
3.The activity of NOS in the penis in the model-moxibustion group was higher than that in the model group (total NOS,cNOS P＜0.01,iNOS P＜0.05) and lower than that in the blank group(total NOS P＜0.05,cNOS,cNOS P＞0.05) after the treatment. The activity of NOS in the model group was much lower than that in the blank group after the treatment (P＜0.01).
4.After the treatment, there was no difference in the concentration of cGMP in the penis between the model-moxibustion group and the blank group (P＞0.05) and the concentration of cGMP in the two groups were much higher than that in the model group (P＜0.01).
5.After the treatment, the expression of RhoA mRNA in the penis in the model group measured with RT-PCR increased significantly, compared with that in the blank group (P＜0.05). The expression of RhoA mRNA in the model-moxibustion group was lower than that in the model group, though there was no difference between the two groups (P＞0.05). And there was no difference in the expression of RhoA mRNA between the model-moxibustion group and the blank group (P＞0.05)
The expression of RhoA protein in the penis measured with immunohistochemistry technique increased significantly in the model-moxibustion group and the model group, compared with that in the blank group (P＜0.01) after the treatment. And the expression of RhoA protein in the model-moxibustion group was much lower than that in the model group (P＜0.01).
The expression of RhoA protein in the penis in the model-moxibustion group and the model group measured with Western blot increased significantly, compared with that in the blank group (P＜0.01) after the treatment. And the expression of RhoA protein in the model-moxibustion group was much lower than that in the model group (P<0.01).
6:After the treatment, the expression of ROCKII mRNA in the penis measured with RT-PCR increased significantly in the model-moxibustion group(P＜0.05) and the model group (P＜0.01), compared with that in the blank group. And the expression of ROCKII mRNA in the model-moxibustion group was lower than that in the model group,though there was no difference between the two groups (P＞0.05)
The expression of ROCKII protein in the penis tissue measured with immunohistochemistry technique increased significantly in the model-moxibustion group and the model group, compared with that in the blank group (P＜0.01) after the treatment. And the expression of ROCKII protein in the model-moxibustion group was lower than that in the model group (P＜0.05).
The expression of ROCKII protein in the penis measured with Western blot increased significantly in the model-moxibustion group(P＜0.05) and the model group (P＜0.01), compared with that in the blank group after the treatment. And the expression of ROCKII mRNA in the model-moxibustion group was lower than that in the model group (P＜0.05).
Conclusions:
1.Moxibustion could decrease the level of blood glucose and promote the erectile function of rats with diabetic ED.
2.Moxibustion could increase the activity of total NOS, cNOS and iNOS and the concentration of cGMP in the penis of rats with diabetic ED, which proved that the NO-cGMP signal pathway might play a role in improving the erectile function when the rats were treated with moxibustion.
3.Moxibustion could decrease the expression of RhoA/Rho kinase in the penis of rats with diabetic ED, which proved that the RhoA/Rho kinase signal pathway might also play a role in improving the erectile function when the rats were treated with moxibustion.
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