Traditional Chinese Medicine for Removing Blood Stasis and Activating Collaterals Improves Renal Inflammation in diabetes Nephropathy Rats by Inhibiting Macrophage Infiltration and Activation through TAK1/JNK Pathway
Diabetes nephropathy (DN) is a common and serious complication of diabetes and the primary cause of end-stage renal disease worldwide. At present, the treatment of DN mainly includes limiting dietary sodium intake, controlling blood sugar, lowering blood pressure, and blocking the renin-angiotensin system (RAS). Although it can delay the progression of DN to a certain extent, its efficacy is not ideal. More and more evidence suggests that inflammatory response plays an important role in the occurrence and development of diabetic nephropathy, and macrophage infiltration and activation are central links in renal inflammation. The TAK1/JNK signaling pathway is an important pathway that regulates macrophage infiltration and activation. Therefore, blocking the TAK1/JNK signaling pathway is an effective strategy for preventing and treating DN.

Our research group believes that “blood stasis obstructing collaterals” is the basic pathogenesis of diabetic nephropathy, and adding traditional Chinese medicine for removing blood stasis and promoting collaterals on the basis of syndrome differentiation and treatment has become the basic principle for treating diabetic nephropathy. The traditional Chinese medicine for removing blood stasis and promoting blood circulation contains numerous drugs. After more than 20 years of clinical practice and repeated screening, our research group has finally determined five Chinese medicines, namely Chuanxiong, Danshen, Dilong, Leech, and Scorpion, as the relatively fixed basic drugs for treating DN. Our previous research has confirmed that traditional Chinese medicine for removing blood stasis and promoting blood circulation can inhibit the activation of renal RAS in DN rats, reduce the loss of podocyte pore membrane protein and cytoskeletal protein, and delay the progression of DN. But there is currently no research on the inflammatory response of this drug in DN state. In view of this, this study takes SD rats as the research object to observe the effects of traditional Chinese medicine for removing blood stasis and promoting collaterals on macrophage infiltration, activation, and TAK1/JNK signaling pathway in renal tissue of DN rats, further clarifying the targets of traditional Chinese medicine for removing blood stasis and promoting collaterals, and providing experimental basis for its clinical application.

In 40 SD rats, 10 rats were randomly selected as the normal group (Group C), and the other 30 rats were fed with a single intraperitoneal injection of streptozotocin (STZ, 40 mg/kg) combined with high sugar and high-fat diet to establish diabetes models. The successfully modeled rats were randomly divided into a model group (M group) and a Chinese herbal medicine group (Z group). Group Z received traditional Chinese medicine intervention. After 16 weeks, 24-hour urine protein quantification (24-hour UTP) was performed in each group of rats; ELISA method was used to detect the expression of monocyte chemoattractant protein-1 (MCP-1), interleukin-1 β (IL-1 β), and tumor necrosis factor – α (TNF – α) in serum; Renal tissue was collected and immunofluorescence was used to detect the expression of macrophage marker protein CD68 and classic activated macrophage marker protein inducible nitric oxide synthase (iNOS). Western blot was used to detect the expression of TAK1/JNK signaling pathway related proteins, including transforming growth factor activated kinase-1 (TAK1), p-TAK1, c-Jun N-terminal kinase (JNK), and p-JNK.

In recent years, traditional Chinese medicine has gained a deeper understanding of diabetic nephropathy, with most medical practitioners arguing based on blood stasis. When suffering from thirst, Qi deficiency and inability to transport blood lead to delayed blood flow, which gradually leads to blood stasis over time; Yin deficiency and excessive fire can cause suffering of body fluids, resulting in insufficient body fluids and poor blood circulation. Over time, this can lead to blood stasis. The “Blood Syndrome Theory: Blood Stasis” states: “When blood stasis occurs between the meridians and organs, it forms the root cause of the disease.” The “Essential Reading of the Medical School” states: “The accumulation of blood stasis is righteousness, accumulated over time, day and night.” If blood stasis persists and does not heal, it is caused by meridian and collateral diseases. The course of DN is lengthy, so its progression conforms to the changes in traditional Chinese medicine, such as aggregation and formation, accumulation over time, and stasis and obstruction of collaterals. The principle of “blood stasis obstructing collaterals” runs through DN from beginning to end and is its fundamental pathogenesis. The method of promoting blood circulation, removing blood stasis, and unblocking collaterals has become the basic treatment principle for DN. However, this blood stasis is difficult to dissipate due to its accumulation, which is beyond the reach of conventional blood activating and stasis removing drugs. Therefore, our research group added leeches, earthworms, scorpions, and other specialized drugs for dredging the meridians and removing parasites on the basis of using conventional blood activating and stasis removing drugs such as Danshen and Chuanxiong. Danshen is an essential medicine for promoting blood circulation and removing blood stasis, with its main active ingredients being salvianolic acids and tanshinones. Modern pharmacological studies have shown that Danshen can regulate glucose and lipid metabolism in DN patients and experimental animals, inhibit oxidative stress, inflammation, and fibrosis, and protect renal function. Chuanxiong has the effect of promoting blood circulation and qi circulation. Its main active ingredients are ligustrazine and ferulic acid. Modern pharmacological studies have shown that Chuanxiong has various biological activities, such as inhibiting platelet aggregation and thrombosis, reducing blood viscosity, decreasing mesangial cell proliferation, and inhibiting endothelial cell proliferation. The efficacy of leeches is to break blood, remove blood stasis, and promote menstruation. The main active ingredient of leeches is hirudin, which has anticoagulant, antithrombotic, lipid-lowering, anti-inflammatory effects, inhibition of epithelial mesenchymal transition, reduction of apoptosis of renal tubular epithelial cells, and alleviation of renal fibrosis. Earthworms have the effects of promoting blood circulation and diuresis in treating kidney disease. Lumbricus contains earthworm kinase, which has the effects of improving the imbalance of the fibrinolytic system, inhibiting platelet activation, regulating lipid metabolism disorders, and inhibiting the proliferation of glomerular mesangial cells and excessive accumulation of extracellular matrix. Scorpion has the effects of dispelling wind, unblocking meridians, and dispersing knots. Modern pharmacological studies have shown that scorpions can anticoagulate, antithrombotic, promote fibrinolysis, increase renal blood flow, and reduce proteinuria. The combination of these five herbs can unblock the meridians and eliminate blood stasis.

Recent studies have shown that macrophage infiltration is one of the characteristic manifestations of DN inflammatory response. Macrophage infiltration was found in both DN patients and DN animal models, and the degree of infiltration was positively correlated with DN proteinuria and renal dysfunction. Research has shown that the main factor determining the direction of kidney disease development is not the amount of macrophage infiltration, but rather the activation status of macrophages in local tissues. After kidney injury, monocytes in the blood migrate into kidney tissue and differentiate into macrophages. Macrophages acquire two different activation phenotypes in a specific microenvironment, namely classically activated macrophages (M1) and non classically activated macrophages (M2). M1 macrophages mediate kidney inflammation and fibrosis by secreting iNOS, chemokines (MCP-1, IL-8), and pro-inflammatory factors (TNF – α, IL-1 β); M2 produces anti-inflammatory factors such as IL-1 and IL-10, which have anti-inflammatory and tissue repair functions. INOS is a marker of M1 macrophage activation. In the early stage of kidney injury, M1 macrophages infiltrate mainly and release inflammatory factors such as MCP-1, IL-1 β, TNF – α, ROS, etc., inducing and enhancing the inflammatory response. In this study, compared with the normal group, the model group of rats showed an increase in the aggregation of CD68 positive macrophages in renal tissue, an increase in the expression of M1 macrophage activation marker iNOS, and an increase in the levels of inflammatory factors such as MCP-1, IL-1 β, TNF – α. Chinese herbal medicine for removing blood stasis and promoting blood circulation can inhibit the infiltration, activation, and release of inflammatory factors of renal macrophages in DN rats, thereby reducing renal inflammation.

The infiltration and activation of macrophages in DN renal tissue are closely related to the TAK1/JNK signaling pathway. JNK belongs to the mitogen activated protein kinase family (MAPK). Each MAPK family is composed of a set of three kinases that act in sequence: MAPKK kinase (MAPKKK), MAPK kinase (MAPKK), and MAPK. Activated MAPKKK can phosphorylate MAPKK and activate it. Activated MAPKK phosphorylates MAPK and activates it. Activated MAPK can be translocated into the nucleus to regulate the transcription of target genes. In recent years, animal and cell experiments have confirmed that the JNK pathway promotes renal inflammatory response. TAK1 is a serine/threonine kinase belonging to the MAPKKK family, located upstream of JNK, and is an important signaling molecule in JNK signal transduction. Activation of the TAK1/JNK signaling pathway can be observed in kidney diseases such as DN, IgA nephropathy, and renal transplant ischemia/reperfusion, and is closely related to the progression of inflammatory response. In UUO mice, inhibition of the TAK1/JNK signaling pathway can reduce macrophage infiltration, inhibit M1 macrophage activation, and lower levels of inflammatory factors. In this study, compared with the normal group, the TAK1/JNK signaling pathway was significantly activated in DN rats. Chinese herbal medicine for removing blood stasis and promoting blood circulation can significantly reduce the expression of TAK1/JNK signaling pathway related proteins and inhibit the activation of the TAK1/JNK signaling pathway.

In summary, our research results indicate that traditional Chinese medicine for removing blood stasis and promoting blood circulation can improve proteinuria and alleviate renal inflammation in DN rats. The mechanism of action may be related to its inhibition of TAK1/JNK signaling pathway mediated macrophage infiltration, activation, and release of inflammatory factors.