Protective effects of Shensuitongzhi formula on intervertebral disc degeneration via downregulation of NF-κB signaling pathway and inflammatory response

Low back pain (LBP) is a common orthopedic disease over the world and has become the greatest cause of disability globally [12], resulting in huge financial and social burdens [13]. About 540 million patients are given the diagnostic code reflective of spinal osteoarthritis. LBP is defined as pain and discomfort located to lumbar region and/or gluteal region, anatomically from the 12th thoracic vertebra to the gluteal sulcus with or without radiating pain [14]. In most cases, it is difficult to identify the specific structural cause of pain [15]. At present, surgery has not have a satisfactory long-term effect despite the main clinical treatment for IDD. Based on this, many studies turn to non-invasive alternatives for IDD [16]. More and more Chinese medicines have been dug that they have an amazing treatment on IDD [17‐20]. Shensuitongzhi formula (SSTZF) is a drug commonly used in clinical treatment for orthopedic diseases such as fracture [21], osteoarthritis [21] and osteoporosis. It has been suggested that IDD shows similarity changing in cartilage with articular cartilage degeneration [20]. At the same time, it has been found that SSTZF can also have a good treatment for IDD [22]. But the exact mechanism has not been clarified, which limits the further development and application of SSTZF.

LBP is closely related to lifestyle and living conditions, such as obesity, poor sleep, stress and smoking [23]. The etiology of LBP remains unclearly. Extensive imaging and histopathology studies have found that lumbar intervertebral disc degeneration (IDD) is an important cause of LBP [24]. Physiologically, the intervertebral disc (IVD), an important part of the spine [25], consists of three major tissues: nucleus pulposus (NP), cartilaginous endplate (CEP) and annulus fibrosus (AF) [26]. The pathogenesis of IDD has not been fully elucidated which is characterized as losing and producing insufficiency of extracellular matrix (ECM) because of the decrease in number and function of NP cells [27, 28]. Type II collagen (Col II) and proteoglycan (PG) are the main components in ECM [16]. In the process of IDD, there are not only an obvious imbalance between the anabolism and catabolism of ECM [11] but also producing a large number of inflammatory factors such as TNF-α and IL-1β which promote the reduction of matrix by directly inhibiting the expression of matrix genes or by increasing the expression of collagen and aggregator lyase [29].

Previous studies have mainly focused on the nucleus pulposus which is the core structure of IVD and shows obvious changes in X-ray, while changes in the cartilage endplate are rarely reported [30, 31]. CEP is a thin layer of hyaline cartilage that not only separates the vertebral body from disc [32, 33], but also residues mechanical forces from intravertebral disc pressure [34]. The CEP is a semi-permeable barrier by which nutrients entering the disc and metabolites expelled. Therefore, CEP degeneration not only changes the mechanical function of CEP, but also affects the bio-transport function of CEP [35, 36]. Chondrocyte apoptosis and endplate calcification are considered as two decisive manifestations of cartilage endplate degeneration [37, 38]. Therefore, protection against CEP degeneration is a potential treatment strategy for maintaining IVD health and preventing spinal disease.

The nuclear factor-κB (NF-κB) signaling pathway plays a key regulatory role in inflammatory response and immune response [31]. The importance of NF-κB signaling pathway in the cause and treatment of IDD has been reported in many studies. For example, Wang et al. [39] found that the activation of NF-κB can promote the overexpression of MMPs and then lead to the over-decomposition of ECM, thus leading to IDD. Meanwhile, NF-κB signaling pathway activation can increase many inflammatory mediators and chemokine expression levels, resulting in a vicious cycle that further accelerates the progression of IDD [9, 30, 40]. Studies using losing function approaches confirmed the importance of NF­κB in IDD [40, 41]. In addition, a large number of studies have found that TCM which has efficacy on tonifying kidney and promoting blood circulation can alleviate IDD through NF-κB pathway [42]. Many studies have focused on the action mechanism of NF-kB in NP [30, 31]. However, the effect of NF-κB signaling on cartilage endplates is unknown.

Based on the above-mentioned studies, we hypothesized that SSTZF could ameliorate the pathological changes in inflammation-associated chondrocytes with potential drug therapy for IDD. In the present study, we evaluated the therapeutic effect of SSTZF on IDD and investigated the potential mechanism of action with in vivo and in vitro experiments.

For the first time, we proof the effect of SSTZF on the progression of IDD in animal models and explore its mechanism preliminarily. Histological and μCT results showed that SSTZF effectively delayed the degradation of cartilage matrix, inhibited the expression of LSI-induced inflammatory factors and protected the cartilaginous endplate calcification and intervertebral disc degeneration of LSI-induced mice. SSTZF was also demonstrated to delay IDD progression by inhibiting NF-κB signaling pathway in vivo and in vitro. These data provides a new and reliable evidence for the clinical treatment of LBP by SSTZF.

SSTZF, a traditional Chinese Herbal formula, has many function on different disease not only orthopedic disease but also internal disease [43].Our previous study had demonstrated that SSTZF may decelerate osteoarthritic cartilage degeneration [44] via reducing the expressional level of MMP13 in cartilage of DMM-induced mice [45]. In addition, it was been reported that had an effective treatment on fracture [21]. The recent results showed that SSTZF modulates NP cell proliferation and ECM remodeling in IDD [30]. As the same results as we has, in our experiment, SSTZF has the also function on IDD through restoring the metabolic balance of ECM. In this study, we used the LSI mouse model to simulate the human IDD process. μCT and ABH staining results were similar to those previously reported [46]. Reduced disc height, vertebral endplate sclerosis and decreased IVD volume were observed in LSI mice. However, mice treated with SSTZF showed the opposite result to the model group at 4 and 8 weeks after LSI surgery, suggesting that SSTZF delays disc degeneration and IDD progression by reducing cartilaginous endplate calcification.

Recent studies have shown that cartilage endplate degeneration is also a key factor in the pathogenesis of IDD [47]. The CEP is a thin layer of hyaluronic cartilage that acts as a semi-permeable barrier which not only delivers nutrients but also be a cushion for mechanical load [33]. In the IDD process, the balance between ECM anabolism which mainly include col2 and catabolism which mainly include Mmp13 is broken [11]. In intervertebral disc degeneration, Mmp13 expression quantity significantly increases in IDD as well as in arthritis [20]. As a result, Col2 expression quantity decreased and Mmp13 expression quantity increased in LSI group. The expression of Col2 and Mmp13 in the SSTZF group was significantly increased compared with LSI group. Therefore, SSTZF can maintain the normal integrity of cartilage endplate by restoring the metabolic balance of ECM, delaying the calcification and degeneration of the endplate.

During the IDD process, a large number of inflammatory factors, such as TNF-α and IL-1β, are produced in ECM. These cytokines promote matrix degradation, chemokines production and cell phenotypic change [48], leading to the dysmetabolism of ECM. In the previous report, inflammatory cytokines are closely related to NF-κB signaling pathway [49]. Also, it has been demonstrated that NF-κB is involved in IDD by regulating ECM degradation, oxidative stress, aging and cell death [31]. NF-κB exists as a dimer which is a member of the Rel protein family. Common Rel proteins are Rel A (p65), RelB, c-­Rel, p50 and p52 [50]. Rel A (p65) is one of the most widespread proteins in eukaryotic cells and has the most vital regulatory roles [51]. Also, phosphorylation and nuclear translocation of P65 are the classical activation of the NF-κB [52]. Therefore, the expression of p-P65 was detected. Compared with the model group, the expression of p-P65 in the SSTZF group was significantly reduced. These results suggest that SSTZF may delay IDD progression by inhibiting the activation of NF-κB. Previous studies have shown that SSTZF has a wide range of favorable therapeutic effects on orthopedic diseases, many of which are achieved through anti-inflammatory and regulation of internal stromal metabolic status. This is the first time that our results demonstrated that SSTZF can reduce inflammatory response and regulates stromal homeostasis in the treatment of IDD through inhibiting the activation of NF-κB.

However, the shortcoming of our present study is that we have not detected the molecular pharmacological mechanism of the active SSTZF. Investigating the possible biological processes of SSTZF may be beneficial for further research and help us using the SSTZF more effectively.

In our study, the degeneration of LSI-induced mice’s tissue phenotype can be salvaged by SSTZF. The function of these, on the one hand, is that SSTZF can inhibit the ECM degradation pathway of intervertebral disc tissue and, on the other hand, is that SSTZF can inhibit the activation of NF-κB. Our results suggested that SSTZF can be used in the clinical as an alternative therapy for IDD.