WANG Yi-fan, GU Yue, SHEN Yu-hang, DING Xin, LYU Zhe, WANG Xin-ai, GUO Deng-zhou

1.The First Affiliated Hospital of Hebei University of Chinese Medicine, Shijiazhuang 050000, China

Keywords:

ABSTRACT Objective:To observe the effect of Jiawei Danggui Buxuetang on the expression of microRNA-155 (miR-155) and cytokine signal transduction inhibitor 1 (SOCS1) in renal tissue of rats with diabetic nephropathy (DKD), and to explore the mechanism of Jiawei Danggui Buxuetang in the treatment of DKD.Methods:Firty 6-week-old SPF male SD rats were randomly divided into normal group (CON) and model group.The model group was fed with high-sugar and high-fat diet for 6 weeks, and the DKD rat model was established by intraperitoneal injection of streptozotocin (STZ) (35 mg/kg).Then the rats were randomly divided into model group (MOD), low dose group (DBTL), medium dose group (DBTM),high dose group (DBTH) and irbesartan group (IRB).Each group was given intragastric administration for 20 weeks, during which CON group and MOD group were given the same amount of normal saline.Urine samples were collected after administration, and the ratio of urinary Microalbumin to urinary creatinine (UACR) was analyzed by automatic analyzer.The renal changes in each group were observed by hematoxylin-eosin (HE) staining, Masson pine (Masson) trichromatic staining and PASM staining.The expression of miR-155 and SOCS1mRNA in renal tissue of DKD rats was detected by Real-time PCR, and the expression of SOCS1 was detected by Western blotting.Results:By comparison with CON group, UACR value, MIR-155 mRNA expression and SOCS1 mRNA expression in MOD group increased significantly, while SOCS1 mRNA expression decreased significantly compared with MOD group, UACR decreased significantly in IRB group and DBT group, SOCS1 expression increased significantly in DBTM group, DBTH group and IRB group.The expression of MIR-155 decreased significantly in all treatment groups (P＜0.05), and the expression of SOCS1 in DBTM group, DBTH group and IRB group increased significantly (P＜0.05).Conclusion:Jiawei Danggui Buxuetang can reduce the expression of miR-155 in high glucose and increase the expression of SOCS1 in renal tissue, so as to significantly reduce renal damage and play a role in renal protection.

1.Introduction

According to statistics, the prevalence of diabetes mellitus (DM),the number of deaths and the medical and health expenditure continue to rise worldwide, and the number of DM patients aged 20-79 is expected to rise to 642 million by 2040[1].Diabetic nephropathy (DKD) is a serious progressive complication of DM,and it is estimated that 20%-40% of DM patients will develop DKD, which is a major cause of end-stage renal disease (ESRD),cardiovascular events, and all-cause death[2].At present, the clinical treatment of DKD is mainly based on the comprehensive intervention of changing lifestyle, controlling blood sugar, blood pressure and correcting metabolic disorders, but its clinical therapeutic effect and prognosis are still not ideal.Therefore, it is still necessary to explore effective prevention and control methods and targets of DKD.

Traditional Chinese medicine has profound theoretical basis and practical experience for the early prevention and clinical diagnosis and treatment of DKD[3, 4].Our team′s previous research has also confirmed [5-8],danggui buxuetang and its flavorful formula can play a multi-channel and multi-target role in anti-oxidative stress,reducing inflammatory response, inhibiting or reversing podocyte scorch death and injury, and delaying the progression of DKD.In recent years, several studies have shown that[9-11], microRNA-155 (miR-155)/cytokine signal transduction inhibitor 1 (SOCS1)axis is involved in regulating the renal response to hyperglycemia and the pathological process of DKD occurrence and development.Its expression in vivo is very stable, and it is expected to become a biomarker and therapeutic target for DKD.Therefore, based on previous studies, this study further explored the effects of Jiawei danggui buxuetang which was established with the basic treatment of supplementing qi and promoting blood circulation, removing blood stasis and clearing collagories, on the expression of miR-155 and SOCS1 in the kidneys of DKD rats.To explore whether Jiawei danggui buxuetang plays a renal protective role by regulating miR-155/SOCS1 axis.

2.material

2.1 Experimental animals and feeding materials

Fifty 6-week-old SPF male SD rats (Beijing Weitonglihua Laboratory Animal Technology Co., LTD.), body weight (200±20)g, experimental animal license No.: SCXK (Beijing) 2016-0011,were fed on conventional diet and high-fat and high-sugar diet in the Laboratory Animal Center of Hebei University of Traditional Chinese Medicine, and drinking water was not limited.This experiment was reviewed and approved by Animal Ethics Committee of Hebei University of Traditional Chinese Medicine, Ethics number:DWLL202206004.

2.2 Drugs and Reagents

Jiawei danggui buxuetang without decocting granules(Astragalus 30 g, Angelica 6 g, Salvia miltiorrhizae 10 g, Dilong 10g,Guangdong Yifang Pharmaceutical Co., LTD., batch number:A1069511, A1069011, A1062271, A1125281), Irbesartan tablets(Sanofi Hangzhou Pharmaceutical Co., LTD., batch number:1348J20026, 0.15 g/ tablet).Streptozotocin (sigma Company, article number: S0130-50MG); Sodium citrate buffer (Sigma-Aldrich, item No.: S4641-25G); Rabbit primary antibody SOCS1(BIOSS, item No.: bs-23696r); BCA Protein Quantitative detection kit for RIPA Lysis solution (Servicebio, item number: G2002) and TBS buffer solution (Servicebio, item number: G0001-2L) (Servicebio, item number: G2026); RNA Extract (Servicebio, part number: G3013)

2.3 Experimental apparatus

One Touch glucose meter (LifeScan); Clinitek50 urine analyzer(Bayer USA); RT-6100 Enzyme marker (Rayto); KZ-II, KZ-Ⅲ-FP grinding instrument (Servicebio); DS-S100 Digital Pendulum Shaker(Servicebio); SVE-2, SVT-2 swimming apparatus (Servicebio);Vortex Mixer (Servicebio); Vertical electrometer (Servicebio);Transfer electrometer (Servicebio); Ultrasonic cell fragmentation Instrument (Xinzhi Biotechnology Company); Fluorescence quantitative PCR instrument (Bio-rad); Table Top High Speed Refrigerated micro centrifuge (DragonLab)

3.Methods

3.1 Preparation of experimental animal models

Fifty 6-week-old male SPF SD rats with body weight of (200±20)g were prepared for routine adaptive feeding.One week later,blood samples were taken from the tail vein to measure the fasting blood glucose in the normal range and the weight was measured.The patients were divided into a normal group (CON) with 8 and a modeling group with 42 using the random number table method.The CON group was given a normal diet, and the modeling group was given a high-sugar and high-fat diet for 6 weeks.Streptozotocin(STZ) was then dissolved in sodium citrate buffer at 0.1 mmol/L(pH=4.5), and the modeling group was injected intritoneally at the dose of 35 mg/kg, while the CON group was given an equal dose of sodium citrate buffer.After STZ injection, the modeling group was given high glycemic water and the normal group was given normal saline.72 h later, blood samples taken from the tail vein for 3 consecutive days for blood glucose ＞ 16.7 mmol/L were used to establish the model successfully.Rats that did not conform to the standard of the synthetic model continued to be intraperitoneally injected with STZ at the dose of 35mg kg-1 as before 3 d later[12].

3.2 Animal grouping, drug intervention, sampling and fixation

In the process of model preparation,2 rats died 1 d and 2 d after STZ injection, and a total of 40 rats successfully modeled were randomly divided into 5 groups.They were divided into model(MOD) group, Jiawei danggui buxuetang low-dose group (DBTL),Jiawei danggui buxuetang medium-dose group (DBTM), Jiawei danggui buxuetang high-dose group (DBTH) and Irbesartan group(IRB), with 8 animals in each group.The administration groups were given irbesartan aqueous solution (0.017 g/kg) and Jiawei danggui buxuetang aqueous solution (2.94 g/kg, 5.88 g/kg, 11.76 g/kg)once a day by intragastric administration according to "human and animal body surface area equivalent dose conversion formula"[13].CON group and MOD group were given the same volume of normal saline intragastric administration, continuous intervention for 20 weeks.After 20 weeks of drug intervention, the rats were deprived of water for 12 h, and the rats were anesthetized by abdominal injection with 3% pentobarbital sodium at the rate of 40 mg/kg.After kidney removal, the left kidney was cut open vertically, and the corresponding cortical tissue of the kidney size was taken and placed in 2.5% glutaraldehyde (pre-cooling) and stored at 4 ℃.The 0.5cm renal cortex tissue was fixed in 4% paraformaldehyde for examination.Part of the renal cortical tissue was stored in liquid nitrogen and sent to the -80 ℃ cryogenic refrigerator for examination.

3.3 UACR values of rat urine samples were analyzed by urine analyzer

After drug intervention, the urine samples of rats were collected,the contents of mALB and Cr were detected by urine analyzer, and the ratio was calculated as UACR value, so as to evaluate the kidney injury of rats in each group.

3.4 HE, Masson and PASM staining were used to observe the pathological changes of DKD rats

After fixed for 48 h, the above specimens were embedded in paraffin, sliced (4 μm) and dewaxed by water.The slices were stained with HE, Masson and PASM, respectively.After dehydration,the slices were sealed, and the pathological morphological changes of the renal tissue specimens were observed with a microscope.

3.5 The expression level of SOCS1 in rat kidney was detected by Western blot

The protein concentration was measured by BCA method.The protein solution was added into 5× reduced protein loading buffer(the ratio was 4:1), and the protein was denatured by boiling water bath for 15 min.Electrophoresis was performed according to SDSRAGE gel electrophoresis instructions.PVDF:300 mA constantflow membrane was used for 30 min.The solution of 5% skim milk powder was closed for 30 min and cleaned with TBST for 3 times.The primary antibody SOCS1 was diluted with TBST at 1:3 000 and slowly shaken overnight in a shaker at 4 ℃.The second antibody is diluted with TBST at 1:5 000 and added to the incubation tank, which is slowly shaken on a shaker and incubated at room temperature for 30min.TBST is an instant-washing film for three times, and then TBST is added and placed on a decolorizing shaker for three times for rapid elute, once every 5 min.Chemiluminescence development.alphaEaseFC software was used for strip gray value analysis, and the relative expression of target protein = target protein gray value /GAPDH gray value.

3.6 The expression levels of SOCS1 and micro-155 mRNA in rat kidneys were detected by Real-time PCR

Rat kidney histopins were extracted with RNA extract solution,and RNA concentration and purity were determined by Nanodrop 2 000.The RNA with excessive concentration was diluted in proper proportion, and then PCR amplification was performed by configuring reverse transcription system.Predenaturation at 95 ℃for 30 s, denaturation at 95 ℃ for 15 s, annealing at 60 ℃ for 30 s,40 cycles.The mRNA relative expression was calculated by 2-ΔΔCt.The primer was designed and synthesized by Wuhan Servicebio technology Company, as shown in Table 1.

Tab 1 Primer sequence of PCR

3.7 Statistical processing

Data, through the analysis of the IBM SPSS 23.0 software measurement data using mean + / - standard deviation(±s) said,inspection accord with normal distribution and variance together,using the single factor analysis of variance (One - wayANOVA); If the above conditions were not met, non-parametric rank sum test was used, and P ＜ 0.05 was considered statistically significant.

4.Results

4.1 Analysis of the effect on the degree of renal injury in rats

Compared with CON group, UACR in MOD group was significantly increased (P＜0.01).Compared with MOD group,UACR in IRB group and DBT group was significantly decreased(P＜0.01), and DBTH group was the most significantly decreased(P＜0.01).See Table 2 and Fig 1.

Fig 1 Modified Danggui Buxuetang on renal tissue injury in DKD rats

Tab 2 Modified Danggui Buxuetang on Renal Tissue Injury in DKD Rats(n=8, ±s)

Tab 2 Modified Danggui Buxuetang on Renal Tissue Injury in DKD Rats(n=8, ±s)

Note: Compared with CON group, P ＜ 0.05; Compared with CON group, P ＜ 0.01; c compared with MOD group, P ＜ 0.05; d compared with MOD group, P ＜ 0.01 (the same below)

Group Dose of medication(g/g) UACR (mg/g)CON - 19.1±4.07 MOD - 128.6±6.62bDBTL 2.94 107.44±9.73dDBTM 5.88 97.81±9.12dDBTH 11.76 72.65±5.66dIRB 0.017 91.15±8.64dF 197.42

4.2 Observation of pathological changes of renal tissue in DKD rats

HE staining results showed that compared with the CON group, the renal tubular epithelial cells in MOD group increased in volume, the cytoplasm was transparent (Figure 2, B long tail arrow), the nucleus was located in the center or to one side, and the glomerular vascular capillaries were dilated, and mononuclear cells were infiltrated.Compared with MOD group, mesangial hyperplasia and renal tubule damage were reduced in DBT and IRB groups.MASSON staining results showed that, compared with CON group, collagen hyperplasia in glomeruli of MOD group was abundant (FIG.3, B long tail arrow), occupying capillary space in vascular bulb.Compared with MOD group, there was no obvious hyperplasia of collagen in the glomeruli of DBTH and IRB groups, but the hyperplasia of collagen in the glomeruli of DBTM and DBTL groups was reduced.PASM staining results showed that compared with CON group, the basal membrane of glomeruli in MOD group was significantly thickened(FIG.4, B long tail arrow), and the capillaries in the vascular bulb were dilated.Compared with MOD group, the glomerular basal membrane thickening in DBTH and IRB groups was significantly reduced, while the basal membrane thickened slightly in DBTM and DBTL groups, forming an obvious double-layer structure (FIG.4, E F long tail arrow).See Figure 2,3,4.

4.3 SOCS1 expression in serum of rats in each group was detected by Western Blot

Compared with MOD group, SOCS1 expression in DBTM group,DBTH group and IRB group was significantly increased (P＜0.01),but there was no statistical significance between DBTL group and MOD group (P＞0.05).See Table 3 and Figure 5.

Fig 5 Jiawei Danggui Buxuetang on SOCS1 protein electrophoresis in renal tissue

Tab 3 Effect of Jiawei Danggui Buxuetang on protein expression of SOCS1 in DKD rats (n=3,±s )

Tab 3 Effect of Jiawei Danggui Buxuetang on protein expression of SOCS1 in DKD rats (n=3,±s )

Group Dose of medication(g/kg) SOCS1/GAPDH MOD - 0.49±0.11 DBTL 2.94 0.77±0.17 DBTM 5.88 1.05±0.3dDBTH 11.76 1.33±0.33dIRB 0.017 1.31±0.15dF 7.36

Fig 3 Jiawei Danggui Buxuetang on renal pathological changes of DKD rats (MASSON,×400)

Fig 4 Jiawei Danggui Buxuetang on renal pathological changes of DKD rats (PASM,×400)

4.4 The mRNA expression of SOCS1 and miRNA-155 in renal tissues was detected by RT-PCR

Compared with CON group, the expression level of MIR-155 in MOD group was significantly increased (P＜0.01), and the expression level of SOCS1 in MOD group was significantly decreased(P＜0.01).Compared with MOD group, the expression level of MIR-155 in all drug administration groups was significantly decreased(P＜0.05 or P＜0.01).DBTH group had the most significant effect(P＜0.05).SOCS1 expression in DBTM, DBTH and IRB groups was significantly increased (P＜0.01).See Table 4.

Tab 4 Danggui Buxuetang on gene expression of miR-155/SOCS1 in DKD rats (n=3,±s )

Tab 4 Danggui Buxuetang on gene expression of miR-155/SOCS1 in DKD rats (n=3,±s )

Group Dose of medication/ g·kg-1miR-155 mRNA SOCS1 mRNA CON - 0.77±0.25 1.04±0.05 MOD - 1.64±0.09b0.47±0.07bDBTL 2.94 1.24±0.14c0.58±0.08 DBTM 5.88 0.88±0.16d0.79±0.06dDBTH 11.76 0.75±0.14d0.88±0.06dIRB 0.017 1.07±0.12d0.85±0.04dF 14.22 35.39

5.Discussion

DKD is a chronic kidney disease caused by hyperglycemia in the body and an independent risk factor for the progression of chronic kidney disease (CKD).Proteinuria is the most important clinical feature.[4,14]In recent years, prevalence rate of DM is increasing obviously [15], The prevalence of DM in adults can reach 11.2%[16],However, 20%-40% of these patients are complicated with DKD[17].Due to the relatively limited therapeutic methods, a large number of DKD patients are still unable to avoid the progression of ESRD, and need to rely on hemodialysis and kidney transplantation to prolong the survival[1].TCM prevention and treatment of DKD has its unique advantages and has attracted more and more attention from scholars at home and abroad in recent years.

Diabetic nephropathy can be classified into several categories,such as "kidney elimination", "hate", "edema", and "urine opacity",etc."On the Syndrome Prescription of Three Causes and One Disease" says, "There are three thirst diseases, namely, quenching thirst, eliminating middle and eliminating kidney." "Shengji Zong Lu" pointed out: "Chronic thirst disease, kidney Qi injury, kidney water, kidney Qi deficiency cold, gasification disorders, unfavorable opening and closing, water accumulation in the body and edema.It reflects that the theory of traditional Chinese medicine has systematically studied and recognized the etiology and pathogenesis of diabetic nephropathy.The etiology and pathogenesis of DKD in the early stage are insufficient Qi and Yin, endogenous heat, long course of disease, Qi deficiency and inability to promote blood transport, blood stasis, blockage of meridians and collaterals,coupled with Qi deficiency and blood stasis, poor operation, loss of viscera in the nourish, eventually resulting in deficiency of Qi and blood Yin and Yang.Therefore, chronic deficiency of body,poor blood circulation and blood stasis are the key factors for the occurrence and development of diabetic nephropathy.Jin Yuan everyone Li Gao′s book "Internal and External injury confusion"recorded in the famous decoction Angelica tonifying blood is the representative of the method of Qi and blood circulation, which astragalus and angelica with 5:1 ratio, in the clinical treatment of atherosclerosis, chronic heart failure, vascular dementia and other diseases has achieved excellent results[18-20].Based on this pathogenesis evolution process, our research group added Danshen Dilong on the basis of Angelica blood-enriching Decoction, with Astragalus membranaceus as the monarch, aiming to replenish Qi and solid surface.Invisible gasification produced tangible blood,Qi was filled with blood, and Angelica was taken as the minister to enhance its ability to produce blood and blood.With Salvia miltiorrhiza promoting blood circulation by stasis, Dilong through the meridian and activating collaterals, the whole formula plays Qi and activating blood circulation, promoting blood circulation by stasis.Pharmacological studies of modern Chinese medicine have shown that astragaloside IV, the active ingredient in Astragalus membranaceus, can control the release of inflammatory factors and reduce oxidative stress damage through various signaling pathways such as PI3K/Akt, NF-κB and JAK/STAT [21]; The bioactive components of angelica sinensis, such as Z-ligustilide and ferulic acid, have significant effects on anti-inflammatory, antioxidant,immunomodulatory and renal protection activities [22]; Earthworm extract can act on PI3K/Akt signaling pathway, inhibit platelet activation and improve microcirculation.Lumbrokinase extract has become a commonly used antithrombotic drug in clinical practice[23]; Tanshinone and salvianolic acid in Salvia miltiorrhiza also have significant effects on anti-myocardial ischemia, anti-platelet aggregation and inhibition of thrombosis [24].The results provided pharmacological evidence for the clinical efficacy of the Jiawei danggui buxuetang decoction.

MicroRNA is a kind of non-coding small molecule RNA with length ranging from 21 to 24 nucleotides.It binds to the target messenger RNA (MRNA) through base complementary pairing to control the translation or degradation of the target MRNA [9].As a member of the microRNA family, miR-155 is widely present in T cells and macrophages.SOCS1, a molecular protein capable of negatively regulating multiple cytokine signal transduction, is one of the important target genes of miR-155.Under the continuous state of high glucose in the body, mitochondria produce a large number of reactive oxygen species, promoting the imbalance of the immune system.miR-155 is widely involved in the response of the human immune system, promoting the mass degradation of SOCS1 mRNA and reducing the expression of SOCS1 by binding with the SOCS1 subregion.SOCS1, as a cytokine signaling inhibitor, can effectively inhibit the activation of signaling pathways such as JAK/STAT and reduce cellular inflammatory response.When miR-155 is overexpressed, the expression of SOCS1 protein is inhibited, and a large number of phosphorylated JAK and STAT3 enter the nucleus to activate downstream inflammatory or apoptotic factors such as BAX and IL-6, which further mediates the occurrence of cellular inflammatory response and apoptosis, and finally accelerates the progression of DKD disease[25-27].In this study, compared with the CON group, pathological changes such as glomerular hypertrophy,mesangial hyperplasia, basal membrane thickening and hyalinization of renal tubular epithelial cells were observed in MOD group under light microscope.UACR value increased significantly.The expression of MIR-155 mRNA was significantly increased, while the expression of SOCS1 mRNA was significantly decreased,suggesting that overexpressed miR-155 was involved in the process of kidney injury in DKD rats.After the intervention of traditional Chinese medicine, the pathological morphology of kidney tissue of rats in each group was improved to varying degrees under the light microscope, and the UACR value was decreased to varying degrees,suggesting that the Jiawei danggui buxuetang has a good renal protection effect.The expression level of miR-155 mRNA decreased and the expression of SOCS1 mRNA and SOCS1 protein increased in the medium and high dose groups of supplemented Jiawei danggui buxuetang, suggesting that this prescription can reduce kidney injury and play a protective role in kidney by regulating the expression of miR-155 and SOCS1.In summary, supplemented angelica blood-enriching soup may reduce the overexpression of miR-155 under the condition of high glucose, reduce the inhibition degree of miR-155 on SOCS1 protein and improve the expression of SOCS1 protein, thus inhibiting the activation of downstream inflammatory or apoptotic factors and preventing the occurrence of cellular inflammatory response or apoptosis, thus alleviating kidney injury.Play renal protective role and delay the progression of DKD.This prescription can be used as a new feasible strategy for TCM clinical prevention and treatment of DKD.

Description of author′s contribution

WANG Yi-fan:Experimental design and implementation, data processing and analysis, paper writing;GU Yue:Participate in experiment design and implementation;SHEN Yuhang:Experimental animal rearing, experimental specimen collection;DING Xin:Experimental animal rearing, experimental specimen collection;LYU Zhe:Technical guidance of laboratory animals;WANG Xin-ai:Data analysis technical guidance;GUO Deng-zhou:Experimental design, experimental content coordination and planning, technical guidance.

Conflict of interest: There is no conflict of interest in this article.