New bitongling (NBTL) ameliorates rheumatoid arthritis in rats through inhibiting JAK2/STAT3 signaling pathway

Abstract

Rheumatoid arthritis (RA) is featured by a variety of physical symptoms and fibroblast-like synoviocytes (FLSs) abnormal proliferation. Increasing evidence has demonstrated that traditional Chinese medicine exerts an important role in RA treatment. New bitongling (NBTL) as one of the traditional Chinese medicine has been reported to be involved in the progression of RA, but the exact mechanism is unclear. In our study, we intended to investigate the effect of NBTL on RA to identify the mechanisms related to JAK2/STAT3 signaling pathway. Extracts of Tripterygium wilfordii (TW), a traditional Chinese herbal medicine, have been widely used for treating RA in China for several decades, so, TW was used as a positive control drug for TBNL. RA rats were constructed by immunization with collagen type II to evaluate the action of NBTL in vivo. Body weight and arthritic index were evaluated. Hematoxylin and Eosin staining was performed to analysis the morphological changes of ankle joints tissue. TUNEL and flow cytometry were performed to examine cell apoptosis, while CCK8 and Ethynyl-2'-deoxyuridine (EdU) were performed to examine cell proliferation. In addition, the markers of inflammation were detected by Western blot, ELISA, and RT-qPCR. Firstly, we find that rats treated with NBTL or TW not only reduced swelling degree and bone destruction, but also repressed IL-1 β and IL-6 levels. In addition, NBTL and TW could increase the weight of rats, and promote the level of IL-10 and IL-4 in vivo. Furthermore, NBTL inhibited inflammation of FLS, induced cell apoptosis and hindered cell proliferation, which was reversed by dipeptidyl peptidase (DPP), a JAK2/STAT3 pathway activator. Taken together, NBTL potentially retarded RA via JAK2/STAT3 pathway, highlighting novel mechanisms associated with RA.

Figure 1.

Inflammation inhibitory effects of NBTL on rheumatoid arthritis (RA) in vivo. RA rats were constructed by immunization with collagen type II, then we treated RA rats with different concentrations of NBTL, and TW (positive control drug for NBTL). A) Observation of the paw swelling. B) Body weight of rats in treated groups. C) Arthritic scores of rats were used to assess the severity of the disease. D) ELISA was performed to analyze the serum levels of IL-1β, IL-6, IL-10, and IL-4. Data were shown as mean ± SD (n = 10 for all groups). ^*p<0.05, ^**p<0.01 vs Control, ^#p<0.05, ^##p<0.01 vs RA.

Figure 2.

NBTL inhibits cell proliferation in RA in vivo. A) Damage to joint tissues determined using H&E staining (200 ×). B) The mRNA expression levels of MCM7, PCNA and Ki-67 were measured by qRT-PCR. C) The protein expression of proliferation-associated proteins MCM7, PCNA and Ki-67 were measured by Western blot. Data were shown as mean ± SD (n=10 for all groups). ^**p<0.01, ^***p<0.001 vs Control, #p<0.05, ^##p<0.01 vs RA.

Figure 3.

The validation and inflammation of fibroblast-like synovial (FLS) cells isolated from RA rats treated with NBTL. A) The expression levels ofv were determined using immunocytochemistry (200 ×). B) The positive cell rate of CD55 were determined using flow cytometry. C) ELISA was performed to analyze the supernatant levels of IL-1β, IL-6, IL-10, and IL-4 from FLS cells. Data were shown as mean ± SD (n=3). ^*p<0.05, ^**p<0.01 vs Control.

Figure 4.

NBTL inhibits cell proliferation and promotes apoptosis of FLS in vitro. A) The cell apoptosis determined using TUNEL staining (400 ×). B) The cell viability was determined using CCK8 assay. C) The cell proliferation was determined using EdU staining (400 ×). D) The cell apoptosis determined using flow cytometry. E) The mRNA expression levels of Bcl-2, Bax, caspase-3 and caspase- 9 were measured by qRT-PCR. F) The expression of apoptosis-associated proteins, including Bcl-2, Bax, Cleaved caspase-3, Cleaved caspase-9 were measured by Western blot. Data were shown as mean ± SD (n=10 for all groups). ^*p<0.05, ^**p<0.01 vs Control.

Figure 5.

The effect of JAK2/STAT3 pathway on NBTL’s impacts on cell proliferation and apoptosis of FLS in vitro. We treated FLS with both NBTL and DPP (JAK2/STAT3 pathway activator) simultaneously. A) The protein expression levels of JAK2/STAT3 pathway were measured by Western blot. B) The cell viability was determined using CCK8 assay. C) The cell proliferation was determined using EdU staining (400 ×). D) The cell apoptosis was determined using the TUNEL assay (400 ×). E) The cell apoptosis was determined using flow cytometry. F) The protein expression levels of apoptosis-associated proteins, including Bcl-2, Bax, Cleaved caspase-3, Cleaved caspase-9, were measured by Western blot. Data were shown as mean ± SD (n=10 for all groups). ^**p<0.01 vs Control, ^#p<0.05, ^##p<0.01 vs C.