| In our previous study, recombinant activating gene-1 (Rag-1-/-) mice, lacking both T and B cells, were used. Male Rag-1-/- mice received adoptive transfer of female or male CD3+ T cells 3 weeks before 14-day Angll infusion.After adoptive transfer of male (CD3M→Rag-1-/--M) or female (CD3F→Rag-1-/--M) T cells, Ang Ⅱ significantly increased blood pressure in CD3M→Rag-1-/--M mice when compared with CD3F→ Rag-1-/--M mice. CD3M→Rag1-/--M mice exhibited higher splenic frequencies of proinflammatory interleukin-17A and tumor necrosis factor-a and lower plasma levels and renal mRNA expression of interleukin-10 than CD3F→Rag-1-/--M mice. The sex of the recipients is the same, so the donor’s T cells differenciation are affected by both sex hormone and sex chromosome of donor mice. In order to explore that the sex differences in hypertension and in T cells differenciation are determined by sex hormone or sex chromosome, we did the next research. Male and female Rag-1-/-mice received adoptive transfer of male CD3+T cells 3 weeks before 14-day AngII infusion. After adoptive transfer of male T cells, Ang Ⅱ significantly increased systolic blood pressure in males (CD3M→Rag-1-/--M) when compared with females (CD3M-→Rag-1-/--F). We came to a conclusion that the sex of the recipient (sex hormone of the recipient) leads to sex differences of T cells differenciation and blood pressure. And estrogen plays an anti-hypertension role.In order to explore the effects of sex hormone and sex chromosome on sex differences in hypertension, and the relationship between sex differences and T cells differenciation in hypertension. Male Rag-1-/- mice received bone marrow (BM) of female or male C57 mice 3 weeks before 14-day AngⅡ infusion. Blood pressue, heart rate and Th, Tc, Tregs proportion in blood and splenocytes of both BMM→Rag-1-/--M mice and BMF→Rag--/--M mice were tested. Gonadectomized male and female SHR rats were used in our study.Exogenous estradiol (E2), Testosterone (T), and Icariin were given to the gonadectomized SHR rats to explore the effects of sex hormone and sex chromosome on the blood pressure and T cells differenciation. In addition, we explored the sex hormone-like effects of Icariin, which provided experimental basis for the application of plant hormones on the treatment of sex-specific hypertension.This study is divided into two parts:literature review and experimental research.1.literature Review:①Sex Hormone and Hypertension ② Sex-Specific T-Cell Regulation of Hypertension ③ Protective Effects of Icariin on Cardiovascular Diseases2. Experimental Research 1:Irradiation dose of Rag-1-/-mice for bone marrow transplantationAim:to explore the irradiation dose of Rag-1-/-mice for bone marrow transplantation, and test the proportion of bone marrow from donor B6 mice in Rag-1-/-mice as a recipient after bone marrow transplantation by flowcytometry.Methods:Rag-1-/-mice were chosen as a recipient, which have CD90.2 on the surface of bone marrow cells, and B6 mice were chosen as a donor, which have CD90.1 on the surface of bone marrow cells. Rag-1-/-mice were irradiated with a dose of 500rads or 800rads. And four hours after irradiation, Rag-1-/-mice were intravenously injected with 5×107 bone marrow cells (BM) isolated from B6 donor mice. Three weeks later, we tested the proportion of CD90.1+peripheral blood mononuclear cells (PBMCs) differentiated from BM in the Rag-1-/- mice by flowcytometry. According to the proportion of CD90.1+PBMCs from donor mice in CD90.2+PBMCs from recipient mice, we can test whether the bone marrow transplantation is successful.Results:①Both Rag-1-/- mice after irradiation of 500rads (Rag-1-/--M+800rads) and Rag-1-/-mice receiving BMafter irradiation of 500rads (BMM→Rag-1-/--M+800rads) survived. Rag-1-/- mice after irradiation of 800rads (Rag-1-/--M+800rads)lost weight anddied within three weeks after transplant while all of the Rag-1-/-mice receiving B6 mice BM after irradiation of 800rads (BMM→Rag-1-/--M+800rads) survived.② Flowcytometry results:Proportion of CD90.2+ PBMCS in Rag-1-/- mice was 96.12±0.83%, and proportion of CD90.1+ PBMCS in B6 mice was 98.85±1.04%. Proportion of CD90.1+PBMCS in BMM→Rag-1-/--M+800rads was only 49.7±0.97%, and proportion of CD90.2+ PBMCS was 50.3±1.11%. Proportion of CD90.1+PBMCS in BMM→Rag-1-/--M+800rads was 93.16±1.53%, above 90%.Conclusion:800rads irradiation irreversibly kills bone marrow cells in Rag--/-mice. After transplant of B6 mice 5×107 BM, the Rag-1-/-mice have above 90% of CD90.1+ PBMCS. The bone marrow transplantation is successful.2. Experimental Research 2:Sex-specific T-cell regulation of hypertension in ratsAim:to explore the role of sex chromosomes in the sex differences of hypertention and in the T cell differentiation.Methods:Male Rag-1-/-mice were irradiated with a dose of 800rads, and four hours after irradiation, they were intravenously injected with 5x107 unfractionated bone marrowcells (BM) isolated from male or female C57 donor mice (BMM→Rag-1-/--M+800rads or BMF→Rag-1-/-M+800rads). Five weeks later, Rag-1-/- mice were given AngⅡ intervention. We tested the survival curve, body weight, mean arterial blood pressure (MAP) and heart rate (HR) of the Rag-1-/-mice. Proportion of T cell subtypes in the blood cells and splenocytes were tested by flowcytometry.Results:①BMF→Rag-1-/--M+800rads and BMM→Rag-1-/--M+800rads survived for 54 days after irradiation, while the Rag-1-/--M mice receiving 800rads irradiation (Rag-1-/--M +800rads) died within 23 days.② After bone marrow transplant, BMM→Rag-1-/--M+800rads mice showed more weight gain than BMF→Rag-1-/---M+800rads mice (P<0.01)③ After AngⅡ intervention, BMF→Rag-1-/--M+800rads showed higher blood pressure than BMM→Rag-1-/--M+800rads mice (P<0.01)④ After AngⅡ intervention, BMF→Rag-1-/--M+800rads mice showed faster heart beats than BMM→Rag-1-/--M+800rads mice (P<0.01)⑤ After AngⅡ intervention, there were no significant differences of Tc and Th proportion in blood cells between BMM→Rag-1-/--M+800rads mice and BMF→Rag-1-/--M+800rads mice (P>0.05). Proportion of Thl7 in blood cells of BMF→Rag-1-/--M+800rads mice was higher than the one in blood cells of BMM→Rag-1-/--M+800rads mice (P<0.01), and compared with the Tregs proportion in blood cells of BMM→Rag-1-/--M+800rads mice, the Tregs proportion in blood cells of BMF→Rag-1-/--M+800rads mice tended to increase (P>0.05) ⑥ After AngⅡ intervention, there were no significant differences of Tc and Th proportion in splenocytes between BMM→Rag-1-/--M +800rads mice and BMF→Rag-1-/--M+800rads mice (P>0.05). Proportion of Th17 in splenocytes of BMF→Rag-1-/--M+800rads mice was higher than the one in splenocytes of BMM→Rag-1-/--M+800rads mice(P<0.01), but there was no significant differences of Tregs proportion in splenocytes between BMM→Rag-1-/--M+800rads mice and BMF→Rag-1-/--M+ 800rads mice (P>0.05)Conclusion:Besides sex hormone, sex chromosome is another factor which leads to sex-specific hypertension. Under the same sex hormone condition, compared with XY chromosome complement, XX chromosome complement leads to a higher proportion of Th17, which is related to AngⅡ-induced hypertension.3. Experimental Research 3:Sex hormone-like protective effects of Icariin on regulation of hypertension in SHRAim:to explore the effects of exogenous sex hormone and the autologous sex chromosome on blood pressure, heart rate, and T cells differentiation, and to investigate the sex hormone-like protective effects of Icariin on regulation of blood pressure in SHR.Methods:Male SHR rats were given orchiectomy (ORX) and female SHR rats were given ovariectomy (OVX). After two weeks, the castrated male SHR mice were given estradiol, testosterone, low dose of Icariin and high dose of Icariin intervention and were divided into 7 groups:WKY-M, SHR-M, SHR-E2-M (E2:0.1mg/kg/day), SHR-T-M (T:26mg/kg/month), SHR-I-M (Icariin:20mg/kg/day), SHR-HI-M (Icariin: 40mg/kg/day), SHR-HI+Flu-M (Icariin:40mg/kg/day, Flutamide:22.3mg/kg/8 hours). And the ovariectomized female SHR mice were given estradiol, testosterone, low dose of Icariin and high dose of Icariin intervention and were divided into 7 groups:WKY-F, SHR-F, SHR-E2-F(E2:0.1mg/kg/day), SHR-T-F(T:26mg/kg/month), SHR-I-F(Icariin:20mg/kg/day), SHR-HI-F(Icariin:40mg/kg/day), SHR-HI+ICI-F (Icariin:40mg/kg/day, Fulvestrant/ICI 182780:52mg/kg/months).We test the body weight, blood pressure, and heart rate of the male and female SHR everyweek. Six weeks after given drugs interventions, all of the rats were sacrificed. We tested the weight of heart, spleen, left kidney, right kidney and uterus (female) and calculated organ coefficients. Proportion of Th, Tc, Tregs in PBMCs and splenocytes were tested by flowcytometry. Serum levels of E2, Testosterone, AngⅡ, TNF-a and IL-17 were tested by Elisa. Aortic arches were isolated for HE and Masson staining. The expressions of ERβ and AR in aorta were tested by Western-blot.Results:①After given testosterone, male SHR rats (SHR-T-M) had significantly body weight gain (P<0.01). After given estradiol, female SHR rats (SHR-E2-F) had significantly body weight loss (P<0.01). However, after given Icariin, there were no significant changes of body weight in both male and female rats (P>0.05)② Treatment of castrated male SHR mice with E2 or Icariin resulted in blood pressure drops (P<00.01), while testosterone elevated the blood pressure (P<0.01) Treatment of ovariectomized female SHR mice with E2 or Icariin resulted in blood pressure drops (P<0.01), while testosterone elevated the blood pressure (P<0.01), Effects of Icarin on blood pressure can be blocked by estrogen receptor antagonist (P<0.01)② Treatment of castrated male SHR mice with testosterone or Icariin resulted in heart rate drops (P<0.01), while E2 had no effects on heart rate (P>0.05). Effects of Icarin on blood pressure can be blocked by androgen receptor antagonist (P<0.01) Treatment of ovariectomized female SHR mice with testosterone or Icariin resulted in heart rate drops (P<0.01), while E2 had no effects on heart rate (P>0.05)③ Treatment of castrated male SHR mice with testosterone resulted in weight gain of both heart and kidney (P<0.01). Treatment of ovariectomized female SHR mice with testosterone resulted in weight gain of heart, kidney, and uterus (P<0.01). Treatment of ovariectomized female SHR mice with E2 or Icariin resulted in weight gain loss of spleen (P<0.01)⑤ Compared with WKY rats, aorta of SHR rats had a higher fibrosis degree. E2 or Icariin treatment was found to attenuate the fibrosis degree of aorta in male castrated SHR rats, and the effects of Icariin can be blocked by androgen receptor antagonist. E2 or Icariin treatment was found to attenuate the fibrosis degree of aorta in female ovariectomized SHR rats, and the effects of Icariin can be blocked by estrogen receptor antagonist.⑥ Flowcytometry results of peripheral blood:Testosterone or Icariin treatment was found to attenuate the proportion of Th cells in peripheral blood cells of male castrated SHR rats (P<0.01), while estradiol had no effects on Th proportion (P>0.05). E2, testosterone and Icariin had no effects on Th proportion in peripheral blood cells of female ovariectomized SHR rats (P>0.05)Testosterone or Icariin treatment was found to attenuate the proportion of Tc cells in peripheral blood cells of male castrated SHR rats (P<0.05), while estradiol had no effects on Tc proportion (P>0.05), and the effects of Icariin can be blocked by androgen receptor antagonist (P<0.05). E2 or Icariin treatment was found to attenuate the proportion of Tc cells in peripheral blood cells of female ovariectomized SHR rats (P<0.05), while testosterone had no effects on Tc proportion (P>0.05)Testosterone or Icariin treatment was found to elevate the proportion of Tregs cells in peripheral blood cells of male castrated SHR rats (P<0.05 or P<0.01), while estradiol had no effects on Tregs proportion (P>0.05). E2 or Icariin treatment was found to elevate the proportion of Tregs cells in peripheral blood cells of female ovariectomized SHR rats(P<0.01), while testosterone had no effects on Tc proportion (P>0.05), and the effects of Icariin can be blocked by estrogen receptor antagonist (P<0.05)⑦ Flowcytometry results of splenocytes:There were no effects of E2, testosterone and Icariin on Th proportion in splenocytes of gonadectomized SHR rats (P>0.05)Testosterone or Icariin treatment was found to attenuate the proportion of Tc cells in splenocytes of male castrated SHR rats (P<0.05), while estradiol had no effects on Tc proportion (P>0.05). E2 or Icariin treatment was found to attenuate the proportion of Tc cells in splenocytes of female ovariectomized SHR rats (P>0.05), and the effects of Icariin can be blocked by androgen receptor antagonist (P<0.05).Icariin treatment was found to elevate the proportion of Tregs cells in splenocytes of male castrated SHR rats (P<0.05), and testosterone had a trend to elevate the proportion of Tregs cells in splenocytes of male castrated SHR rats (P>0.05), while estradiol had no effects on Tregs proportion (P>0.05). And the effects of Icariin can be blocked by androgen receptor antagonist (P<0.05). E2 or Icariin treatment was found to elevate the proportion of Tregs cells in splenocytes of female ovariectomized SHR rats (P<0.01 或 P<0.05), while testosterone had no effects on Tregs proportion (P>0.05), and the effects of Icariin can be blocked by estrogen receptor antagonist (P<0.05)E2, Testosterone or Icariin treatment was found to attenuate the content of TNF-a in splenic T cells of male castrated SHR rats (P<0.01). E2 or Icariin treatment was found to attenuate the content of TNF-a in splenic T cells of female ovariectomized SHR rats (P<0.05), while testosterone had no effects on content of TNF-a (P>0.05). E2 or Icariin treatment had no effect on content of IL-17 in splenic T cells of male castrated SHR rats (P>0.05), while testosterone was found to elevate the content of IL-17 (P<0.01). E2, testosterone or Icariin treatment was found to attenuate the content of IL-17 in splenic T cells of female ovariectomized SHR rats (P<0.01) (9) E2 treatment was found to elevate the level of serum E2 and attenuate the content of AngⅡ, TNF-a and IL-17 in serum of both male and female SHR rats (P<0.01), and testosterone was found to elevate the level of serum testosterone, Angll, TNF-a and IL-17 in serum of both male and female SHR rats (P<0.01). Icariin was found to attenuate the level of serum TNF-a and IL-17 in serum of both male and female SHR rats (P<0.01), while had no effects on the levels of serum AngⅡ, E2 and testosterone, and the effects of Icariin can be partly blocked by estrogen receptor antagonist and androgen receptor antagonist (P<0.01) ⑩ E2 treatment had a trend to elevate the expression of ERβin aorta of male castrated SHR rats (P>0.05), while testosterone had no effect on the expression of ERβ (P>0.05), and Icariin was found to attenuate the expression of ERβ (P<0.01).E2 or testosterone had a trend to elevate the expression of ERβ in aorta of female ovariectomized SHR rats (P>0.05), while Icariin had no effect on expression of ERβ (P>0.05)E2 or Icariin treatment was found to attenuate the expression of AR in aorta of male castrated SHR rats (P<0.01), while testosterone had no effect on the expression of AR (P>0.05). E2, testosterone or Icariin was found to attenuate the expression of AR in aorta of female ovariectomized SHR rats (P<0.01)Conclusion:1. E2 is found to attenuate blood pressure of female ovariectomized SHR rats, which is related to the role of E2 that attenuates the Tc proportion, Angll level and fibrosis of aorta and elevates the Tregs proportion. E2 is found to attenuate blood pressure of male castrated SHR rats, which has no relation to the regulation of T cells differentiation.2. Testosterone is found to elevate blood pressure, but attenuate heart rate of male and female SHR rats, which is related to the role of testosterone that elevates Angll level and fibrosis of aorta. In addition, the effects of testosterone on blood pressure have no relation to the regulation of T cells differentiation.3. Icariin itself can not elevate the serum of sex hormone and expression of sex hormone receptor. Icariin is found to attenuate heart rate and blood pressure of gonadectomized SHR rats, which is related to the role of Icariin that attenuates the Tc proportion, serum Angll level, serum TNF-α, serum IL-17 and fibrosis of aorta and elevates the Tregs proportion. The effects of Icariin can be partly blocked by estrogen receptor antagonist and androgen receptor antagonist. Icarin has sex hormone-like effects. |
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