Mechanism Of Cistanche Shaoyao Powder In Alleviating Inflammatory Responses In Diabetic Nephropathy By Modulating Macrophage Polarisation In The Kidneys Of Db/Db Mice
Nov 07, 2025
HOU Luyu¹, ZHENG Linlin², SHI Wenjing¹, WANG Zixuan¹, GUO Shilong¹, LYU Zhe¹², GUO Dengzhou¹²³*
¹ Graduate School, Hebei University of Chinese Medicine, Shijiazhuang 050000, China
² The First Affiliated Hospital of Hebei University of Chinese Medicine, Shijiazhuang 050000, China
³ Hebei Provincial Technology Innovation Center for Traditional Chinese Medicine Treatment of Spleen and Renal Diseases, Shijiazhuang 050000, China
Abstract
Objective: This study aims to investigate the effect of Cistanche Shaoyao Powder-a traditional herbal formula consisting of Cistanche deserticola, Paeoniae Radix Alba, Atractylodes macrocephala, Chuanxiong Rhizoma, Poria cocos, and Alisma orientalis-on macrophage polarisation and inflammatory responses in the kidneys of db/db mice with diabetic kidney disease (DKD), and to explore its renoprotective mechanisms.
Methods: Eight db/m mice were designated as the normal control group. Forty db/db mice were randomly divided into five groups: model group, low-dose (8.39 g·kg⁻¹), medium-dose (16.77 g·kg⁻¹), and high-dose (33.54 g·kg⁻¹) Cistanche Shaoyao Powder groups, and an irbesartan group (0.025 g·kg⁻¹). All treatments were administered once daily for 12 weeks. General health conditions were monitored. At the end of the intervention, 24-hour urine samples were collected using metabolic cages. Mice were anesthetised, blood was collected via enucleation, and serum was harvested for measurement of glycated serum protein (GSP), serum creatinine (Scr), blood urea nitrogen (BUN), total cholesterol (TC), and triglycerides (TG). Urinary albumin-to-creatinine ratio (UACR) was measured. Kidney histopathology was assessed by HE, PAS, and Masson's staining. ELISA was used to detect serum TNF-α, IL-10, and MCP-1 levels. Immunofluorescence (IF) was performed to detect F4/80 expression in renal tissues, and immunohistochemistry (IHC) was used to evaluate CD206 expression. Real-time PCR was conducted to measure TNF-α, IL-10, iNOS, and Arg-1 mRNA expression. Western blot analysis was used to detect iNOS, Arg-1, CD86, and CD206 protein expression.
Results: Compared to the normal group, db/db mice in the model group showed significantly increased levels of GSP, UACR, Scr, BUN, TC, TG, TNF-α, and MCP-1, and decreased IL-10 levels (P<0.01). Histological analysis revealed glomerular hypertrophy, mesangial expansion, inflammatory infiltration, tubular vacuolisation, glycogen deposition, and collagen accumulation. F4/80 fluorescence intensity was increased, while CD206 expression was reduced. TNF-α and iNOS mRNA levels were elevated, IL-10 and Arg-1 mRNA levels were decreased. Protein levels of iNOS and CD86 were upregulated, while Arg-1 and CD206 were downregulated (P<0.05, P<0.01). Treatment with Cistanche Shaoyao Powder or irbesartan significantly reversed these changes, reducing serum and urinary markers, inflammatory factors, and histopathological damage, and promoting M2 macrophage polarisation.
Conclusion: Cistanche Shaoyao Powder improves renal function and mitigates pathological kidney damage in db/db mice. Its mechanism may involve inhibiting M1-type macrophage polarisation, promoting M2-type polarisation, reducing inflammation, delaying renal fibrosis, and exerting a renoprotective effect.
Keywords: Cistanche Shaoyao Powder; diabetic kidney disease; macrophage polarisation; inflammation
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Introduction
Diabetic kidney disease (DKD) is a microvascular complication arising from metabolic disorders associated with diabetes. In recent years, the prevalence of diabetes in China has increased from 10.9% in 2013 to 12.4% in 2018–2019. Among these, the prevalence of type 2 diabetes mellitus (T2DM) combined with chronic kidney disease (CKD) is as high as 36% [1]. DKD is a multifactorial disease involving complex interactions between haemodynamic abnormalities and metabolic dysregulation. Key pathogenic drivers include chronic hyperglycaemia, accumulation of advanced glycation end-products (AGEs), and overactivation of the renin-angiotensin-aldosterone system (RAAS) [2].
Clinically, DKD is characterised by a progressive increase in urinary albumin excretion and a steady decline in glomerular filtration rate (GFR), which may eventually progress to end-stage renal disease (ESRD). Treatment of ESRD relies heavily on renal replacement therapy, significantly reducing patients' quality of life and placing a heavy economic burden on healthcare systems. Moreover, DKD remains challenging to manage due to delayed diagnosis, individual differences in treatment response, and poor long-term prognosis.
Chronic inflammation plays a pivotal role in the pathogenesis of DKD [3], with macrophage-mediated inflammatory responses being a critical contributor [4]. Although DKD is not traditionally classified as an immune disease, multiple studies have confirmed the accumulation of macrophages in the glomeruli and interstitium of DKD kidneys, with infiltration levels positively correlating with the rate of renal function decline [7]. Macrophage recruitment and activation can occur in early stages of db/db mouse kidney injury [8], contributing to glomerular and tubular damage, albuminuria, elevated creatinine, fibrosis, and upregulation of chemokines [9].
Under hyperglycaemia and AGE stimulation, podocytes, mesangial cells, and tubular epithelial cells release adhesion molecules, cytokines, and chemokines that recruit and activate macrophages [10], leading to inflammation, mesangial proliferation [11], and progressive renal fibrosis [12].
In Traditional Chinese Medicine (TCM), DKD falls under the categories of "Xiaoke disease" (wasting-thirst syndrome) and "turbid urine". Its pathogenesis involves spleen-kidney deficiency at the root, and water-dampness with blood stasis as the manifestation. Cistanche Shaoyao Powder, derived from Zhang Zhongjing's Jin Gui Yao Lue, is a classic formula with the effects of nourishing blood, invigorating the spleen, and promoting diuresis. The 2024 Guidelines for Integrated TCM and Western Medicine Diagnosis and Treatment of DKD identify blood stasis and dampness as central pathological factors at all stages of the disease [13], thus validating the clinical principle of "invigorating blood and promoting diuresis."
Cistanche Shaoyao Powder, as a representative formula of this principle, has been clinically shown to reduce proteinuria, regulate glucose and lipid metabolism, and improve symptoms in DKD patients [14–16]. However, mechanistic research has largely focused on its inhibition of NF-κB and downstream anti-inflammatory pathways [17,18], with little exploration into its immunomodulatory effects on macrophage M1/M2 polarisation-a key link between inflammation and fibrosis in DKD.
Given the similarities between db/db mice and human DKD, particularly in terms of leptin receptor deficiency, insulin resistance, hyperglycaemia, and macrophage accumulation in the kidneys [19], this study employs db/db mice to investigate how Cistanche Shaoyao Powder affects macrophage polarisation and renal inflammation, aiming to elucidate its molecular mechanisms and provide experimental evidence for clinical application.
