| Objective: The study was designed to elucidate the immunoregulatory effects of Bushen Hemai(BSHM)granules in improving renal damage in ageing spontaneously hypertensive rats,and to study the relationship between Th17 cell/ Tregs balance and hypertensive renal damage from the perspective of adaptive immunity.We porposed the hypothesis that BSHM granules which is a well poved prescription in clinic could alleviate renal damage in ageing spontaneously hypertensive rats(SHRs)by regulating the balance of Th17cell/ Tregs.We hope to provid a reliable laboratory basis for the clinical application of BSHM granules to improve renal damage in ageing hypertensive patients.Methods:1.Clinical research part: Evaluation of the therapeutic effect of BSHM granules on the ageing hypertensive renal damage of kidney qi deficiency.We included the ageing hypertensive renal damage of kidney qi deficiency who were treated in the Department of Cardiology and Geriatrics of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine,from October 2019 to October 2021.The patients who took valsartan and amlodipine tablets continuously for 2 months were considered as the control group,and the patients who took valsartan and amlodipine tablets in combination with BSHM granules continuously for 2 months were considered as the observation group.According to the quantitative tables of Traditional Chinese Medicine symptom grading,we scientifically evaluated the syndrome efficacy of the patients.The blood pressure level and renal function(evaluated by urinary microalbumin,albumin creatinine ratio and glomerular filtration rate)of patients were statistically analyzed to clarify the curative effect of BSHM granules on ageing hypertensive renal damage of kidney qi deficiency.2.In vivo experimental studyExperiment 1: Effects of BSHM granules on blood pressure and adaptive immunity in ageing SHRs.Twenty 18 months old SHRs,SPF level,were randomly divided into two groups(n = 10): model control group(SHR)and Bu Shen He Mai group(BSHM).Ten 18 month old Wistar Kyoto rats were used as the blank control group(WKY).BSHM group was treated with BSHM granules transgastriclly for 8 weeks.The body weight,blood pressure and urine biochemical indexes were recorded to evaluate the overall effect of BSHM granules in vivo;Renal artery blood flow was detected by renal ultrasound;HE staining and transmission electron microscopy were used to observe the histomorphology of kidney and spleen;The degree of renal fibrosis was assessed by Masson staining;Serum level of Interferon-γ(IFN-γ),Interleukin-4(IL-4),Interleukin-6(IL-6),Interleukin-10(IL-10)and Interleukin-17A(IL-17A)was detected by ELISA;The orphanreceptor-γt(RORγt)and Fork-head transcription factor p3(Foxp3)density in spleen were observed by immunofluorescence staining;The amount and proportion of Th17 lymphocytes and Treg lymphocytes in peripheral blood mononuclear cells were detected by flow cytometry;Transcriptome sequencing were used to screen the target m RNAs of BSHM granules in improving hypertensive renal damage.Experiment 2: The mechanism of BSHM granules was explored in reducing hypertensive renal damage,based on Th17 cell/ Tregs balance.Sixty 18 months old SHRs,SPF level,were randomly divided into 6 groups(n = 10): Bu Shen He Mai group(BSHM),SHR group,BSHM+RORγt agonist(SR0987,used to upregulate IL-17 level)group(BSHM granules+7.14mg/kg SR0987,single intraperitoneal injection),BSHM+thiocyanate(S6281,the main source of thiocyanate anion,used to upregulate the IL-10 level)group(BSHM granules+4.4mg/kg S6281,4.4mg/kg S6281,one intraperitoneal injection/2d,a total of 8 weeks),Only SR0987 group(SR0987,7.14mg/kg,single intraperitoneal injection)and Only S6281 group(S6281,4.4mg/kg,one intraperitoneal injection/2d,a total of 8 weeks).Ten age-matched Wistar Kyoto rats of SPF grade were used as the blank control group(WKY).The body weight,systolic pressure,diastolic pressure and urine biochemical indexes were recorded to evaluate the overall effects of BSHM granules in vivo;Renal artery blood flow was detected by renal ultrasound;HE staining was used to evaluate the histomorphology of kidney and spleen;The degree of renal fibrosis was assessed by Masson staining;Serum IL-6,IL-10 and IL-17 A were detected by ELISA assays;Density of RORγt and Foxp3 in spleen were observed by immunofluorescence staining;Quantificational reverse transcriptionpolymerase chain reaction(q RT-PCR)was used to detect m RNA expression level of IL-6,SMAD3,STAT3,Nuclear Factor kappa-B(NF-κB)and transforming growth factor-β1(TGF-β1).The protein expression level of IL-6,SMAD3,STAT3,NF-κB and TGF-β1 were detected by Western blot(WB)analysis.Results:1.Clinical research:1.1 After 2 months of treatments,patients in both control group(valsartan and amlodipine tablets)and the observation group(valsartan and amlodipine tablets+BSHM granules)had gained clinical benefits in reducing blood pressure,improving renal function and quantitative score of Traditional Chinese Medicine syndromes compared to those in their first visit(P< 0.01).1.2 After 2 months of treatments,the blood pressure level,renal function status and quantitative score of Traditional Chinese Medicine syndromes of the observation group(valsartan and amlodipine tablets+BSHM granules)were significantly better than those of the control group(valsartan and amlodipine tablets),the difference was statistically significant(P<0.01).2.In vivo experimental study:2.1 Antihypertensive effect of BSHM granules on ageing spontaneously hypertensive rats BSHM granules administration for 8 weeks effectively decreased systolic pressure by21.2 mmHg and diastolic pressure by 8.8 mmHg(P< 0.01).2.2 BSHM granules improves renal structure and function in ageing SHRBSHM granules reduced urinary microalbumin(m ALB),Beta-2-microglobulin(β2-Mg)and N-acetyl-β-glucosaminidase(NAG)levels of SHRs(P< 0.01).And renal ultrasound results showed significant improvement in renal artery blood flow and arterial stiffness: Peak systolic blood flow Velocity(Vp)and Mean blood flow velocity(Vm)(P< 0.01),Minimum diastolic blood flow Velocity(Vd),Resistance Index(RI)and Pulsatility Index(PI)(P< 0.05);HE staining and transmission electron microscope showed that BSHM granules significantly improved the histological morphology of kidneys of SHRs;Masson staining showed that BSHM granules effectively decreased the area of renal fibrosis(P< 0.01).2.3 BSHM granules inhibits inflammatory response and regulates Th17 cell / Tregs balanceHE staining showed that BSHM granules improved the tissue morphology of spleen of SHRs;ELISA results showed that after 8 weeks’ BSHM granules administration,the level of IL-10(P< 0.05)and IL-6 was significantly decreased(P< 0.01)and IL-17 A was increased(P< 0.05);Although IFN-γ in SHR serum had a downward trend,IL-4 had an upward trend,there was no statistical difference(P>0.05),suggesting that BSHM granules tended to regulate the differentiation and function of Th17 cells / Tregs.Immunofluorescence staining showed that BSHM granules treatment significantly increased the density of Foxp3(P< 0.01)and decreased the density of RORγt(P< 0.01)in spleens of SHRs,suggesting that BSHM granules had the potential to upregulate Treg lymphocytes and downregulate the differentiation of Th17 lymphocytes.The results of flow cytometry showed that BSHM granules significantly reduced the amount and proportion of Th17 cells(P< 0.01)and increased the amount and proportion of Tregs(P< 0.01)in peripheral blood,which confirmed that BSHM granules effectively up-regulated Treg lymphocytes and down regulated the differentiation of Th17 lymphocytes.2.4 Screening drug targets of BSHM granules by transcriptome sequencingTranscriptome sequencing screened 747 differentially expressed genes(m RNAs)in kidney after BSHM granules intervention,of which 418 were down-regulated and 329 were up-regulated.Go analysis suggested the immunomodulatory effect of BSHM granules.2.5 Mechanism exploration of BSHM granules on regulating Th17 cell / Tregs balanceBSHM granules significantly decreased the blood pressure and improved the renal function,blood flow,arteriosclerosis,histomorphology and fibrosis of ageing SHRs.When BSHM granules was used in combination with S6281 upregulating IL-10,the efficacy of BSHM granules in reducing blood pressure and improving renal function was significantly enhanced;When BSHM granules was used in combin combination with SR0987 upregulating IL-17,it was observed that the effects of BSHM granules in reducing blood pressure and improving renal function were offset to some extent.The results of q RT-PCR and WB assays showed that BSHM granules significantly decreased the m RNA and protein expressions of IL-6,STAT3,TGF-β1,NF-κB and SMAD3;When combination with S6281,the m RNA and protein expression of IL-6,STAT3,TGF-β1,NF-κB and SMAD3 were further downregulated;while when BSHM granules combined use with SR0987,the effects were blocked(P < 0.01).Conclusion: BSHM granules reduces the blood pressure level of ageing hypertensive renal damage of kidney qi deficiency type,improved renal function and clinical symptoms.Pharmacological study demonstrated that BSHM granules attenuated hypertensive renal damage in ageing SHRs,improved renal structure,arterial blood flow,arteriosclerosis and tissue fibrosis,alleviated inflammatory responseat least partly,through increasing Treg lymphocytes and decreasing Th17 lymphocytes. |
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