Effect of Total Flavonoids of Oxytropis falcata Bunge on the Expression of p-JAK1-and p-STAT1-Related Proteins in Idiopathic Pulmonary Fibrosis

Objective The study aimed to explore the effect of total flavonoids of Oxytropis falcata Bunge (FOFB) on the expression of p-JAK1/p-STAT1 and SOCS3 proteins in idiopathic pulmonary fibrosis (IPF). Methods Rats model with IPF was established by one-off intratracheal injection of bleomycin (BLM, 5 mg/kg). After 14 days, the same volume of low dose (100 mg/kg), medium dose (200 mg/kg), and high dose (400 mg/kg) of FOFB and prednisolone acetate (20 mg/kg) as positive control drugs, as well as normal saline, were orally administered to rats once a day for 28 consecutive days. Subsequently, the degree of fibrosis and alveolitis in rat lung tissue was observed, respectively, by HE and Masson staining. Further more, observing the ultrastructure of lung tissue by transmission electron microscopy (TEM), the detection of JAK/STAT pathway related indicators including p-JAK1, p-STAT1, and SOCS3 with immunohistochemistry and SOCS3 with real-time PCR (RT-PCR) was performed. Results Compared with the BLM group, the degree of alveolitis and fibrosis improved significantly, and the expression of p-JAK1 and p-STAT1 decreased; conversely, the expression of SOCS3 increased in the treatment group. Conclusion IPF causes high expression of p-JAK1 and p-STAT1 and low expression of SOCS3. FOFB can play a role in the treatment of IPF via upregulating SOCS3 and downregulating p-JAK1 and p-STAT1.


Introduction
Idiopathic pulmonary fibrosis (IPF), characterized by progressive dyspnea, is a chronic progressive interstitial lung disease [1]. After diagnosis, without a lung transplant, the median survival rate is 3-5 years, and the patient eventually dies from respiratory failure [2]. Nowadays, the specific etiology and pathogen are still unknown [3]. Most studies suggest that the occurrence of IPF is characterized by repeated subclinical damage to the alveolar epithelium [4][5][6]. After the lung tissue is damaged, the alveolar epithelium cell is activated. On the one hand, chemokines are released to recruit fibroblasts (FB) and a large number of FB subsequently can produce extracellular matrix (ECM). Besides, FB can also be transformed into myofibroblasts (MFB) under the induction of fibrogenic factors (such as transforming growth factor-β) generating ECM [7]. e excessive deposition of ECM accelerates the occurrence of pulmonary fibrosis. Several key signaling pathways play important role in the regulation process, including JAK/STAT, NF/ κB, and MAPK signaling pathways [8]. Among them, the JAK/STAT signal pathway is most closely related to the occurrence of IPF [9].
JAK protein kinas has a molecular weight of 120-130 kDa. So far, four members, JAK1, JAK2, JAK3, and TYK2, have been identified, which play important and diverse functions in hematopoietic, inflammatory, and immune responses. Among them, JAK1 is the most expressed JAK kinase, which is related to the signal transduction process of many cytokines and involved in inflammation and immune deficiency. STATs have a molecular weight between 79 and 113 kDa, and members of seven STAT protein families including STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6 have been found. STAT1 is the earliest discovered and cloned member of the STAT family [10]. Animal experiments have shown that STAT1 is abnormally expressed in bleomycin-(BLM-) induced IPF mice [11]. JAK1 is inactive before being exposed to cytokines, but the binding of cytokines to their receptors induces their own activation through transphosphorylation (i.e., formation of p-JAK1). Activated p-JAK1 phosphorylates its downstream signaling protein STAT1 (i.e., forming p-STAT1), causing them to dissociate from the receptor and transport to the nucleus. en, p-STAT1 acts as a transcription factor in the nucleus to induce transcription of the target gene [12]. e SOCS protein family (especially SOCS3) is the most widely known negative regulator of the JAK/STATpathway [13]. In addition, SOCS3 also has the ability to directly inhibit JAK kinase activity [14].
Total flavonoids of Oxytropis falcata Bunge (FOFB), extracts of Oxytropis falcata Bunge, are the major active components. Studies have confirmed that it has multiple pharmacologic actions, including being antitumor, being antioxidant, improvement of insulin resistance, protection against UV damage, enhancement of immune cofunction, being antiinflammatory, and analgesia [15,16]. Among them, the antiinflammatory effect is the most significant. Other studies have shown that FOFB can reduce the expression of profibrosis factors such as typeIand type III collagen, inhibit TGF-β1induced human renal tubular epithelial cell proliferation, and   [17,18]. Inflammation plays an irreplaceable role in the process of IPF, and FOFB has a significant anti-inflammatory effect. However, whether FOFB can exert IPF-preventing effects by affecting the expression of inflammatory factors, such as p-JAK1/p-STAT1 and SOCS3, is still unclear.
In the current study, to clarify the specific effects of FOFB on the Inflammation related protein including p-JAK1, p-STAT1, and SOCS3, rats models with IPF were established with BLM. is study aims to find specific targets that block the JAK-STAT signaling pathway in inflammation and provide an experimental basis for the treatment of IPF.

Animals.
Sixty adult specific pathogen-free animal (SPF) Sprague Dawley (SD) rats in half genders (200 ± 20 g) were provided by the Experimental Animal Center of Gansu University of Chinese Medicine. e environmental temperature was controlled at 25 ± 2°C, humidity was maintained at 45%-55%, and circadian rhythm was 12:12 h dark/light. All rats were fed with sterilized diet and water. Animal welfare was in compliance with the principles and procedures of Health Guide for the Care and Use of Laboratory Animals. e study protocols were approved by the Animal Care and Use Committee of Gansu University of Chinese Medicine.

Chemicals and Reagents. Oxytropis falcata
Bunge was purchased from Tianzhu County Tibetan Medicine Development Institute. Total flavonoids of Oxytropis falcata Bunge (FOFB) were extracted and identified by Lanzhou University. e extraction method was ethanol immersion. e specific process included putting 5000 grams of medicinal materials in a 5000 mL round-bottom flask, immersing in 10 times the amount of absolute ethanol, soaking for 3 days, repeating 3 times, taking the filtrate, and spindrying to obtain the product. Bleomycin was obtained from

Grouping and Medications.
Rats were randomly divided into six groups including blank (control) group, model (BLM) group, low-dose FOFB (100 mg/kg) group, middledose FOFB (200 mg/kg) group, high-dose FOFB (400 mg/kg) group, and prednisone acetate group (20 mg/kg) with 10 rats in each group. After intraperitoneal anesthesia with 3% pentobarbital sodium (30 mg/kg), the rats were fixed in supine position. With the help of laryngoscope, BLM (5 mg/ kg) was injected into the trachea. e control group was given the same volume of physiological saline. After the injection, the animal was straightened and immediately rotated left and right to distribute the lung fluid evenly. After 14 days of modeling, rats in each group were given 2 mL of corresponding dose of drugs by gavage once daily. All rats were sacrificed on day 28.

Histopathology.
After sacrifice, part of the left lung tissue was fixed in 4% paraformaldehyde, subsequently embedded in paraffin, serially sectioned, and stained with hematoxylin and eosin (H&E) or Masson's trichrome staining kit (Sigma-Aldrich) following manufacture's protocol. Histopathologic score was graded by specialized histopathologists blindly referring to Szapiel's semiquantitative grading system to divide into 4 grades and described the fibrosis score. e slices stained by Masson's trichrome were picked-up pictures by camera of microscope system and processed by Image-Pro plus version 6.0 for Windows.

Transmission Electron Microscope.
Parts of lung tissue specimen was fixed by 2.5% glutaraldehyde, followed by 1% osmic acid postfixation, routinely dehydrated, and embedded in epoxy resin. It was then sliced into ultrathin sections and observed by a transmission electron microscope and images by AMT camera system were saved.  e reaction conditions were as follows: 95°C denaturation for 5 min, 95°C denaturation for 10 s, 60°C annealing for 20 s, 72°C extension for 20 s, and 42 cycles.

Statistical Analysis.
All data conforming to normal distribution were presented as mean ± standard deviation (SD), and those not conforming to normal distribution were represented by median and interquartile spacing. Data were analyzed by one-way analysis of variance (ANOVA) of the SPSS 24.0 software. Statistical difference was considered significant when p value is less than 0.05.

FOFB Ameliorated the Progress of Inflammation and Fibrosis in BLM-Induced IPF in Rats.
We assessed the extent of inflammation in the lung tissue with HE staining. As shown in Figure 1. e picture of control group showed the bronchial and alveolar structures were intact, the alveolar septum was not thickened, and occasionally inflammatory cell infiltration was observed under the microscope. Sample of BLM group shows most of the alveolar structures collapsed and fused, and the interstitial cells increased, showing obvious fibrosis; the alveolar septa were significantly widened and thickened, and the infiltration of inflammatory cells increased significantly. Compared with the BLM group, low-dose FOFB group showed no significant difference; middle-/high-dose FOFB group and prednisone group could improve the destruction of Evidence-Based Complementary and Alternative Medicine alveolar structure and reduce the area of alveolar space and inflammatory cell infiltration, especially in the FOFB highdose group and prednisone acetate group. Szapiel score is shown in Table 1. e treatment group with middle-/high-dose FOFB and prednisone groups were lower than BLM group (p < 0.05).
We evaluate the degree of fibrosis of the lung tissue by Masson's trichrome, as shown in Figure 2. In the blank group, there was no obvious change in the lung tissue of the rats. e collagen layer of the bronchial wall is thin, and a small number of light blue collagen fibers were distributed in the alveolar wall. In the BLM group, a large number of blue, cord-like, and lamellar collagen fibers stained around the bronchus and small blood vessels and in the lung interstitium were diffusely distributed. And there were a large number of alveolar fusions. e area of collagen deposition was decreased in the middle-/high-dose of FOFB and prednisone acetate groups, especially in the latter two groups.
According to the scoring standard of Masson staining, the results are shown in Table 2.
ere was a significant difference between the highest score of the BLM group and the lowest score of high-dose FOFB and prednisone acetate group (p < 0.01).

FOFB Alleviated the Abnormalities of Lamellar Number
and Structure in IPF Rats Induced by BLM. Lamellar bodies (LBs) are organelles present in type II alveolar epithelial cells (AECII) and are closely related to the secretion of alveolar surfactant. Specific lamellar bodies are the most direct method to identify whether the secretory function of type AECII is normal. In our experiments, as shown in Figure 3, it was observed that the LBs of the blank group had a normal structure, with a large number, and neatly arranged; while the number of model groups was significantly reduced, the structure was incomplete and the arrangement was disordered. Compared with the BLM group, the number of LBs in the treatment with FOFB and prednisone acetate groups increased, the structure tended to be normal, and the arrangement tended to be neat.

Effects of FOFB on the Expression of p-JAK1, p-STAT1, and SOCS3 with IPF in Rats.
e activated p-JAK1/p-STAT1 protein is closely related to the occurrence of inflammation and studies have shown that these two proteins are also abnormal expression in IPF. SOCS3 is the most recognized negative regulator of this pathway. As shown in Figures 4-6, in the control group, immunostaining could be observed in tracheal and vascular wall, and interstitial area was exceedingly weakly positive. In contrast, SOCS3 staining in these sites was strongly positive. Compared with the blank group, in the BLM group, immunostaining of p-JAK and p-STAT1 was strongly positive (p < 0.01); SOCS3 staining was weakly positive (p < 0.01).
e positive rate expression of each treatment group was between the above two groups, and there was no significant difference between the FOFB high-dose group and the prednisone acetate group and the normal group (Figure 7).

Effects of FOFB on the Expression of SOCS3 with IPF in
Rats. RT-PCR reflected that the mRNA expression of SOCS3 was significantly lower in the lung tissue of the model group than that of the blank group (p < 0.05). e results are shown in Figure 8.

Discussion
IPF is an interstitial lung disease with high mortality and poor prognosis [19]. Researchers have found that inflammation was closely related to its occurrence and development [8,20]. JAK1/STAT1 is a key signaling pathway to regulate inflammation [8]. At the same time, studies have shown that JAK1 and STAT1 are abnormally expressed in the lung tissue of IPF mice [8]. SOCS3 is a negative regulator of JAK1/STAT1 and can block the conduction of this signal   [13]. In recent years, Traditional Chinese Medicine plays an important role in the treatment of IPF with its unique advantages and efficacy [21]. Oxytropis falcata Bunge is one of the three anti-inflammatory drugs of Tibetan medicine, among which flavonoids extract has the strongest anti-inflammatory activity [15]. It has been confirmed that FOFB can reduce the expression of some profibrotic factors and slow down the process of renal interstitial fibrosis [18]. Based on the above theory, we investigated the effect of FOFB on the inflammation-induced lung tissue damage, the degree of fibrosis, and the expression of p-JAK1, p-STAT1, and SOCS3. Intratracheal injection of BLM to establish the IPF model is the most classic method because it is similar to the pathological process of human pulmonary fibrosis [22]. In our study, observing the pathological morphology of lung tissue of BLM-induced IPF rats by HE and Masson staining, we found FOFB group could improve the degree of alveolitis and delay the progress of IPF. e existing research has confirmed that p-JAK1 and p-STAT1 were involved in the formation of IPF. JAK1 is inactive before exposure to cytokines, but the binding of cytokines to their receptors induces their own activation through transphosphorylation. Upon activation, phosphorylated JAK1, named p-JAK1, serve as docking sites for members of the STAT transcription factor family. Receptor-localized STAT1 is then phosphorylated by p-JAK1, named p-STAT1, causing a biochemical cascade [14].
rough the results of immunohistochemistry and RT-PCR, we found that the FOFB group could increase the expression of SOCS3 protein and further reduce the expression of p-JAK1 and p-STAT1 to play a therapeutic role.

Conclusion
FOFB has a therapeutic effect on inflammation-mediated idiopathic pulmonary fibrosis, and its mechanism may be to inhibit the expression of p-JAK1 and p-STAT1 inflammatory proteins by upregulating the expression of SOCS3.

Data Availability
All the data related to this article are described as histopathological pictures and statistical analysis in the manuscript.
Ethical Approval e protocol of the experiment was approved by the Ethical Committee for the Experimental Animals at Gansu University of Traditional Chinese Medicine (Gansu, China) (Approval no. 0001391).

Conflicts of Interest
e authors have declared that no conflicts of interest exist.