Anti-inflammatory effects of Jingshu Keli capsule and its components on human synoviocyte MH7A cells

Background Jingshu Keli (JSKL), a traditional Chinese medicine (TCM) formula consisting of multiple active compounds, has been officially approved by National Medical Products Administration (NMPA) for treatment of cervical radiculopathy. It relieves pain, according to TCM theory, by activating blood circulation to dissipate blood stasis. The pain mainly stems from neurogenic inflammation caused by mechanical compression of the cervical nerve root. In addition, inflammation mediators also cause the development of other joint diseases, such as osteoarthritis (OA). The purpose of this paper was to evaluate the anti-inflammatory effects of JSKL and identify the biologically active herbs and compounds in vitro. Methods Enzyme-linked immunosorbent assay (Elisa) was used to determine the expression of pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and interleukin 8 (IL-8), in the culture medium of human MH7A cells stimulated by lipopolysaccharides (LPS). Results JSKL and three single-herb capsules, Cinnamomum cassia Presl (C.C.), Angelica Sinensis (Oliv.) Diels (A.S.) and Carthamus tinctorius L. (C.T.), significantly inhibited the secretion of TNF-α. If one of these three herbal components was removed, suppressing effect of the single-herb-deleted JSKL on TNF-α was abolished. Cinnamaldehyde (CIN) from C.C. was the most potent ingredient that inhibited the expression of IL-6 and IL-8 in the culture medium of both LPS-stimulated MH7A cells and primary synovial cells. Conclusions JSKL was found to possess anti-inflammatory effect in vitro; C.C., A.S. and C.T. were the principal and essential herbal components responsible for such activity; CIN from C.C. is one the most potent single compound among indicator components of JSKL recorded in 2015 Chinese pharmacopoeia. This study provided scientific evidence for the clinical application of JSKL as an agent for targeted treatment of cervical radiculopathy. Furthermore, CIN has potential to be used for the treatment of some inflammation-related orthopedic diseases, such as rheumatic arthritis and osteoarthritis.


Background
Cervical radiculopathy stems from degenerative disease in the cervical spine and seriously affects the quality of life of patients with upper limb pain or motor dysfunctions [1,2]. It is characterized by circumferential narrowing of the cervical foramen and subsequent neural compression [3]. During the process of cervical radiculopathy, inflammation mediators were released [4,5]. Neurogenic inflammation caused by mechanical compression of the cervical nerve root was reported to be the main cause of spastic pain [6][7][8]. Thus, anti-inflammatory analgesics are commonly used for the treatment of arthropathy, such as non-steroidal anti-inflammatory drugs and steroids [9,10]. In addition, the inflammation mediators released can also cause the development of other joint diseases, such as osteoarthritis (OA). Inflammatory molecules secreted by the inflammatory synovium are key mediators of the disruptive processes related to arthritis pathophysiology. Interleukin (IL) and tumor necrosis factor (TNF) control the degeneration of articular cartilage matrix, and, therefore, are the main targets of therapeutic interventions [11]. TNFα and IL-6 appear to be the main pro-inflammatory cytokines involved in the pathophysiology of OA [12,13]. The current treatments for joint diseases mainly focus on the pain control and joint function improvement.
MH7A cells, a human rheumatoid arthritis synovial cell line, exhibit characteristics of inflammatory cells and are often used for establishing RA cell models induced by lipopolysaccharide (LPS) [14,15]. LPS is a component of the cell wall of Gram-negative bacteria [16]. LPS can stimulate MH7A cells to produce pro-inflammatory cytokines, such as TNF-α, interleukin 6 (IL-6) and interleukin 8 (IL-8) [17][18][19]. The Schematic of technology roadmap for assessing the anti-inflammatory effects of JSKL and its compounds in vitro is illustrated in Fig. 1.
Jingshu Keli (JSKL) is one of the commonly used drugs for the treatment of cervical spondylotic radiculopathy in China. It consists of seven traditional Chinese medicines, Panax Notoginseng  [20]. Some clinical validity data have been accumulating [21], and our previous preclinical studies found that JSKL attenuated C7 spinal nerve ligation-induced mechanical allodynia in rats by suppressing the activation of spinal microglia and p-Stat3 [22]. The aim of this study was to confirm the anti-inflammatory effect of JSKL and identify the contributing herbal components and compounds in it by examining the effects of JSKL, single-herb capsules, single-herb-deleted JSKL capsules and reference compounds for quality control in JSKL, as indicated by 2015 Chinese pharmacopoeia, on the expressions of TNF-α, IL-6 and IL-8 in the culture medium of LPSinduced MH7A cells.

Sample preparation
JSKL, single-herb-deleted capsules and single-herb capsules were prepared by using the following methods. Into a 2.5 L four-necked flask equipped with an overhead bladed stirrer, distiller, and thermometer, 66.6 g A.S., 66.6 g L.C., 44.4 g C.C. and 1.5 L distilled water were added. The mixture was heated to the boiling point of water and then heated for 4 h to obtain volatile oil. The volatile oil was encapsulated with 4.2 g β-cyclodextrin. The remaining mixture in the flask was filtered using a 120-mesh filter to obtain Chinese herbal water 1 and dregs 1 and stored separately. 66.6 g P.N., 66.6 g G.E. and 1.0 L distilled water were added into the same flask. The mixture was heated for 1 h and then filtered using a 120-mesh filter to obtain a mixture of Chinese herbal water 2 and dreg 2 and stored separately. The dregs 1, dregs 2 and C.T. were mixed and heated 2 times, with the first time, water was added 3 times and the sample was cooked for 1 h, and with the second time, water was added 3 times and the sample was cooked for 0.5 h. After cooled to room temperature, the mixture was filtered through a 120-mesh filter to get Chinese herbal water 3. The Chinese herbal water 1, 2 and 3 were mingled and the resultant sample was concentrated to a relative density of 1.30 (at 50 to 60°C) using a rotary evaporator. Finally, Bovis Calculus Artifactus (18.5 g), volatile oil and dextrin were added to make a final sample. After drying and pulverizing, a compound sample of JSKL (about 200 g) was obtained. With the aforementioned method, a Chinese herbal medicine was removed in the preparation process to obtain single-herb-deleted capsules. With single-herb capsules, only one Chinese herbal medicine was added. The above-mentioned TCM granules were prepared by Sinopharm Group Jingfang Pharmaceutical Co., Ltd., Anhui, China.

Sample dilution
One milligram sample was dissolved in 0.5 mL water. After mixing, 0.5 mL dimethyl sulfoxide (DMSO) was added. Upon centrifugation, supernatant was taken as stock solution. The monomer compounds were dissolved in DMSO to obtain 10 mmol/L stock solutions.

CCK-8 assay
MH7A cells (4000 cells/well) were seeded into 96-well plates for 12 h and then treated with various doses of samples (JSKL, single-herb-deleted capsules, single-herb capsules and monomer compounds). The cells were incubated in 1640 medium with 10% FBS and 1% P/S for another 48 h. Then, Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Kyushu, Japan) was added into each well and the plates were incubated at 37°C for 2 h, according to the manufacturer's operating instructions. The plates were placed onto an ELISA reader (Bio-Rad, LabWrench, Canada) and the optical density (OD) value of each well was analyzed at 450 nm.

ELISA analysis
MH7A cells (5 × 10 5 cells/well) were seeded into 6-well plates and cultured for 24 h. Then, the cells were treated by various doses of samples for 2 h and induced by 10 ng/mL LPS for 24 h. The cell culture supernatant was collected. The concentrations of TNF-α, IL-6 and IL-8 in the culture supernatant were determined by using the corresponding Human ELISA Kits. The OD value was measured at 450 nm. Then, the relative expressions (RE) of TNF-α, IL-6 and IL-8 were calculated, respectively, according to the following formula: where, OD (treatment) is the OD value of the various doses of samples treatment group; and OD (control) is the OD value of the DMSO control group.
Real-time polymerase chain reaction (real-time PCR) MH7A cells or primary synovial cells were pretreated with of CIN for 2 h, and then incubated for another 6 h with 10 ng/mL LPS. Total RNAs were isolated using the commercial total RNA miniprep kit (Axygen, USA), according to the manufacturer's instructions. Each sample was reversely transcribed using the cDNA synthesis kit (TaKaRa, China), by following the manufacturer's protocol. The primer sequences were used for real-time PCR as shown in Table 1. Real-time PCR was performed using SYBR Green PCR Premix Ex Taq II reagents (TaKaRa) on a Light Cycler 480 II real-time system (Roche, USA), with GAPDH serving as house-keeping gene for normalization [25].

Statistical analysis
All data were expressed as means ± standard derivations (SD) from three independent experiments. Statistical significance between groups was analyzed by employing ANOVA of the GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was set at p < 0.05.

Safe concentrations of JSKL samples on MH7A cells
In order to determine the safe concentrations of JSKL, single-herb capsules, single-herb-deleted capsules on MH7A cells, CCK-8 assay was used to detect the cytotoxicity of these samples at different concentrations. As shown in Table 2, JSKL with concentrations of 0.02 and 0.2 μg/mL could promote the proliferation of MH7A cells, compared with the control group. JSKL and singleherb-deleted capsules showed no cytotoxicity at given concentrations. So, the maximum safe concentration of JSKL and single-herb-deleted capsules was 2 μg/mL. When the concentration of C.C. was 2 μg/mL, the Other herbs (C.T., A.S., L.C., G.E., P.N.) showed no cytotoxicity at any given concentrations. So, their concentrations were set at 2 μg/mL in the following studies. As shown in Table 3     We also investigated the anti-inflammatory effects of CIN in primary synovial cells. The results (Fig. 7b) showed that LPS obviously stimulated the secretions of IL-6 and IL-8 in primary synovial cells.

Discussion
JSKL is one of the TCM herbal medicines for the treatment of cervical spondylotic radiculopathy. In previous studies, we established a rat model of cervical spondylotic radiculopathy by C7 spinal nerve ligation (SNL) with 6-0 silk suture. We found that JSKL could inhibit SNLinduced allodynia and microglia activation [22]. In this study, MH7A cells were used to confirm that JSKL was able to inhibit expression of the main pro-inflammatory factor TNF-α, and three herbal components (C.C., C.T. and A.S.) played essential roles in the action. Further studies demonstrated that CIN from C.C. was the most potent single compound responsible for suppressing LPS-induced and TNF-α-induced expressions of IL-6 and IL-8 in MH7A cells.
TNF-α is a pro-inflammatory cytokine involved in cellular activation and inflammation, and is mainly found in neuromembrane cells and vascular endothelial cells [26][27][28]. Because neuromembrane cells are closely connected with nerve fibers, it is believed that TNF-α may be related to neurogenic pain [29]. The current study, for the first time, clarified that JSKL could inhibit the expression of TNF-α in LPS-induced MH7A cells.
This study examined the effects of JSKL, single-herb and single-herb-deleted capsules on the expression of pro-inflammatory cytokines in MH7A cells. It was found that JSKL could inhibit the expression of TNF-α, while those without C.C., C.T., A.S. and L.C. could not reduce the expression of TNF-α. At the same time, single herbal component, i.e., C.C., C.T., A.S. and L.C. could also inhibit the expression of TNF-α, indicating that C.C., C.T. and A.S. are the main components of JSKL that inhibit the expression of TNF-α. Similarly, JSKL did not affect the expression of IL-6, while JSKL containing no C.C., C.T. and A.S., could increase the expression of IL-6, suggesting that C.C., C.T. and A.S. are important medicinal components or ingredients. On the basis of the findings, we conclude that C.C., C.T. and A.S. are the main antiinflammatory herbs of JSKL.
In this study, we found that C.T. inhibited the expression of TNF-α and IL-8 in MH7A cells. It has been reported that saffower yellower, the main component of C.T., has anti-inflammatory effects [30]. Saffower yellower was also shown to reduce the levels of TNF-α, IL-6, IL-1β and COX-2, inhibit the expression of NF-κΒ P65 protein and NF-κΒ P65 gene, and possess antiinflammatory effect in rats with radicular lumbar spondylosis [31]. A.S. can also inhibit the expression of TNF-α in MH7A cells. Pang et al. [32] established rat inflammation model by intraperitoneal injection of LPS and found that water-soluble low molecular substances in A.S. could significantly reduce the levels of TNF-α and IL-6 in plasma. Ferulic acid, an indicator component of A.S., showed no anti-inflammatory activity at the determined concentration, suggesting that other active compounds might play an anti-inflammatory role in A.S.. CIN, the main component of C.C., significantly decreased the expression of pro-inflammatory cytokines TNF-α, IL-6 and IL-8. Zhang et al. [33] found that CIN could significantly inhibit the expression of prostaglandin E2 and nitric oxide, down-regulate the expression of membrane-related prostaglandin synthase-1 and cyclooxygenase in RAW264.7 cells stimulated by LPS, and had obvious anti-inflammatory and antipyretic effects. The findings suggest that C.C. is an important antiinflammatory TCM herbal component that can be included in the formulation of JSKL capsule, and cinnamaldehyde, the monomer component, was identified to be the key active ingredient for its anti-inflammatory action.
In this paper, CCK-8 assay showed that the safe concentration of JSKL was up to 2 μg/mL with MH7A cells. One limitation of the study is that no data were provided on its in vivo safety. In a previous study, we found that JSKL at the dose of 400 mg/day/ rat attenuated C7 spinal nerve ligation (SNL)-induced mechanical allodynia in rats, without obvious sideeffects after 14-day treatment [22]. Furthermore, the bioactive compound CIN at the dose of 75 mg/kg/day alleviated collagen-induced arthritis in rats, without conspicuous side-effects after a 21-day treatment [34]. Future studies with longer treatment time and organspecific analysis are warranted to evaluate the longterm safety of JSKL.

Conclusions
This study demonstrated that JSKL had antiinflammatory effect in vitro; C.C., A.S. and C.T. were the main and essential herbal components in JSKL responsible for this action; CIN from C.C. was one the most potent single compounds among indicator components specified in 2015 Chinese pharmacopoeia. This in vitro study provided solid evidence that JSKL can be clinically used for prevention and treatment of inflammatory disorders. Furthermore, CIN has potential to be used for the treatment of some inflammation-related orthopedic diseases, such as rheumatic arthritis and osteoarthritis.