Ruscogenin Ameliorated Sjögren's Syndrome by Inhibiting NLRP3 Inflammasome Activation

This article investigated the role and the specific mechanism of Ruscogenin in Sjögren's syndrome (SS). NOD/ShiLtJ mice were treated with Ruscogenin, and acinar cells isolated from submandibular glands were treated with TNF-α, Ruscogenin and transfected with NLRP3 overexpression plasmid. Salivary flow rate (SFR) was measured at weeks 11, 13, 15, 17, and 20. Histological analysis of the submandibular glands was conducted by hematoxylin-eosin staining assay. IL-6, IL-17, TNF-α, and IL-1β mRNA expression was detected through qRT-PCR. AQP 5, AQP 4, P2X7R, NLRP3, caspase 1, IL-1β, Bax, and Bcl-2 protein levels were tested by western blot. Cell apoptosis was assessed through acridine orange and propidium iodide (AO/PI) staining assay and flow cytometry assay. Ruscogenin ameliorated the SFR and submandibular gland inflammation of NOD/ShiLtJ mice. Ruscogenin promoted the preservation of acinar cells and suppressed inflammation-related factors (P2X7R, NLRP3, caspase 1, and IL-1β) in submandibular gland tissues of NOD/ShiLtJ mice. Ruscogenin inhibited acinar cell apoptosis in NOD/ShiLtJ mice and reversed TNF-α-induced apoptosis and inflammation of acinar cells. NLRP3 overexpression reversed the repressive effect of Ruscogenin on TNF-α-induced inflammation and apoptosis of acinar cells. Ruscogenin ameliorated SS by inhibiting NLRP3 inflammasome activation.


Introduction
As the prevalence ranges from 0.1% to 0.72% among the population around the world with a female preponderance ( e ratio of female to male is 9 : 1), Sjögren's syndrome (SS), usually classified as the primary (occurs by itself) and the secondary (relates to other autoimmune diseases like rheumatoid arthritis or lupus), is an autoimmune disorder distinguished by exocrine gland (typically lachrymal and salivary glands) dysfunction owing to infiltration of lymphocytes, leading to excessive dry eyes (keratoconjunctivitis sicca) and dry mouth (xerostomia) [1][2][3][4]. In general, the immune system targets epithelial tissues, then infiltrates with them with lymphocytes, and finally generates autoantibodies against antigens of glands [2]. What's worse, SS is able to develop from disease limited to the exocrine glands to diverse extraglandular manifestations including chronic fatigue, arthralgias, nonerosive polyarthritis, vasculitis, and even lymphoma [5,6]. And with the slow progression of SS, patients with the disease are more likely to perform clinical symptoms years after the disease onset [7]. Without effective treatment of SS, the current SS interventions such as saliva substitutes and medications (cevimeline, pilocarpine, etc.) are symptomatic-based, which can only offer a temporary relief of dryness severity and complications, resulting in economic, physical, and mental burdens on patients with SS [2,8,9]. Hence, it is of urgent significance to search for novel agents with favorable efficacy for SS therapy.
Although the causes of SS remain largely unknown, the inflammation is a well-defined condition among all cases with SS, which can partly attribute to aberrant innate and adaptive immune reactions implicated in pathogenesis of SS [10][11][12]. Besides, the hyposalivation and glandular destruction induced by chronic inflammation during SS are considered to be associated with the injury of acinar cells [13,14]. Inflammasomes are a class of multimeric protein complexes fulfilling a critical function on an innate immune system, the activation of which promotes maturation and secretion of pro-inflammatory cytokines and modulates caspase-1 for further inflammatory events and apoptosis [11,15]. Former studies have shown that nucleotide binding oligomerization domain-like receptor 3 (NLRP3) inflammasome is activated in patients with SS [11], which suggested that NLRP3 inflammasome may act as a therapeutic target for SS management.
Ophiopogon japonicas is a common traditional Chinese herbal medicine mainly consisting of polysaccharides, saponins, and homoisoflavonoidal compounds, whose clinical efficacy like antithrombotic activity and anti-inflammatory property has been validated by scientific evidence [16][17][18]. Moreover, Ophiopogon japonicas is theoretically regarded to realize an effect of "moisturize dryness and facilitate to produce body fluid" and has been utilized in the treatment of xerosis-related dysfunction with traditional Chinese medicine for a long time [16]. As a major bioactive steroid sapogenin isolated from Ophiopogon japonicas, Ruscogenin has been extensively applied in the treatment for chronic inflammation and cardiovascular diseases [18,19]. Additionally, its anti-inflammatory and antithrombotic activities greatly contribute to the improvement of mouse neutrophil activation, cerebral ischemic injury, and pulmonary arterial hypertension [20][21][22]. Nevertheless, the effects of Ruscogenin on SS are largely unknown. Previous studies have reported that Ruscogenin can mitigate blood-brain barrier dysfunction induced by cerebral ischemia through the inhibition of the MAPK pathway and TXNIP/NLRP3 inflammasome [23], and MDG-1 (also named Ophiopogon japonicas polysaccharides), another component of Ophiopogon japonicas, has been confirmed to alleviate symptoms of SS [16].
ose findings suggest that Ruscogenin may regulate NLRP3 inflammasome to impact upon SS. erefore, this study attempted to explore the role of Ruscogenin in SS as well as the specific mechanisms involved through in vivo and in vitro experiments on a basis of NLRP3 inflammasome.

Animals.
Female NOD/ShiLtJ mice aged 8 weeks [2] (n � 40, weighed 18-22 g) were bought from e Jackson Laboratory (Bar Harbor, ME, USA), while female BALB/c mice (n � 10, aged 8 weeks, weighed 18-22 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) as a control. All the mice were allowed free access to food and water, and maintained under specific pathogen-free conditions as well as a relative humidity of 50 ± 20%, an ambient temperature of 22 ± 3°C, and a constant cycle of 12-h light/dark.

Drug Preparation.
Extracted from the tuber of Ophiopogon japonicus, Ruscogenin was purified by successive chromatographic steps until the purity of the sample analyzed using high-performance liquid chromatographyevaporative light scattering detection (HPLC-ELSD) reached 98.6% [23].

Drug Treatment.
Ruscogenin was dissolved in sterile phosphate buffer saline (PBS; C0221A, Beyotime Biotechnology, Shanghai, China). Treatments were administered orally every day, lasting for 9 weeks. NOD/ShiLtJ mice were randomized into four groups (n � 10 per group). e NOD/ ShiLtJ group was normally reared without any treatment; the vehicle group was administered vehicle (an equal volume of sterile PBS); the Ruscogenin 0.3 group was treated with Ruscogenin at 0.3 mg/kg body weight; and the Ruscogenin 1 group was treated with Ruscogenin at 1 mg/kg body weight. (SFR). NOD/ShiLtJ mice were anesthetized by isoflurane (2%) inhalation, following which they were injected intraperitoneally with pilocarpine (C11H16N2O2; 1538505, Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 5 mg/kg for the stimulation of salivary secretion. Saliva was then collected for 7 min using a 200 μL pipette (SE4-200XLS+, Mettler Toledo, Zurich, Switzerland, https://www.mt.com/cn/zh/ home.html), which was adopted to evaluate the total volume. SFR is performed in microliters per gram of body weight per min (μL/gm/min).

Histological
Analysis. Histological analysis of the submandibular gland was performed at week 20 after NOD/ ShiLtJ mice were sacrificed via euthanasia using 100 mg/kg ketamine in combination with 5 mg/kg xylazine. e submandibular glands removed from NOD/ShiLtJ mice were fixed in 10% neutral buffered formalin (G2161, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 24 h, and dehydrated and embedded in wax. Serial sections of the tissues at a thickness of 5 µm were taken through an automated microtome (Leica RM2235, Leica Microsystems, Wetzlar, Hesse-Darmstadt, Germany, https://www.leicamicrosystems.com.cn/cn/). Hematoxylin-Eosin (HE) Staining Kit (G1120, Beijing Solarbio Science & Technology Co., Ltd., China) was utilized for tissue stain. In brief, after deparaffinization in xylene (C8H10, ≥75.0%; 214736, Sigma-Aldrich, USA) twice for 5 min and hydration in 100% ethanol for 5 min, 95% ethanol for 2 min, 80% ethanol for 2 min, and 70% ethanol for 2 min, as well as distilled water for 2 min, the sections were immersed in hematoxylin solution for 5 min and rinsed by water. Sections were then differentiated by differentiation liquid for 30 s, followed by immersion in water for 15 min. Subsequently, sections were immersed in 0.6% ammonia water for 15 min to make the nucleus return back to blue and washed with water. Next, eosin solution was applied to stain sections for 2 min and rinsed with water. Sections were quickly dehydrated in 75% ethanol for 3 s, 85% ethanol for 3 s, 95% ethanol for 3 s, 100% ethanol for 3 s, and 100% ethanol for 1 min, made to be transparent in phenol (C6H6O, ≥99.0%; P1037, Sigma-Aldrich, USA) and xylene (1 : 3) for 1 min and xylene twice for 1 min, and eventually Biomedical Technology Co., Ltd., China). e primer sequences (Guangzhou RiboBio Co., Ltd., Guangzhou, China) are presented in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the endogenous control, and data were calculated via the 2-ΔΔCTrelative quantification method [24].

Isolation of Acinar Cells in Submandibular Gland.
Submandibular glands were quickly removed and immediately transferred to ice-cold Roswell Park Memorial Institute (RPMI)-1640 Medium (E600028, Sangon Biotech Co., Ltd., Shanghai, China) enriched with 10% fetal bovine serum (FBS; 11011-8611, Beijing Solarbio Science & Technology Co., Ltd., China). en, the tissues were minced into small pieces, followed by digestion in 2.5 ml RPMI-1640 medium supplemented with 0.1 g/L soybean trypsin inhibitor (17075029, ermo Fisher Scientific, Waltham, MA, USA), 10% FBS, and 100 U/ml Collagenase IV (17104019, ermo Fisher Scientific, USA) in a shaking water bath at a speed of 120 cycles/min for 10 min at 37°C. At last, the mixture was dispersed using a plastic pipette and filtered through a 150-mesh nylon net. e acinar cells extracted from submandibular glands of female BALB/c mice were taken as a control.

Acridine Orange and Propidium Iodide (AO/PI) Staining
Assay. AO/PI Staining Apoptosis Detection Kit (BB-4142-1, Bestbio, Shanghai, China, http://www.bestbio.com.cn/) was utilized to test cell apoptosis. Reagent C (100 μL) was mixed with sterile deionized water (900 μL) to make staining binding buffer. Cells (1 × 10 6 ) were washed twice with PBS and resuspended in 500 μL staining binding buffer. AO staining solution (5 μL) and PI staining solution (5 μL) were added to cells in turn and mixed gently, following which cells were incubated at 4°C in a dark room for 20 min. Cells were then rinsed by PBS and observed using the Leica DM IL LED microscope (magnification: ×200).

Flow Cytometry Assay. Cell apoptosis was detected by
Annexin V-FITC/PI Apoptosis Detection Kit (E606336, Sangon Biotech Co., Ltd., China). Cells were rinsed with PBS and suspended in 1 × binding buffer (195 μL) at a density of 2 × 10 5 cells/mL. Next, 5 μL Annexin V-FITC was added and mixed well in a dark room, followed by culture for 15 min at room temperature. en, cells were washed by 1 × binding buffer (200 μL) and centrifuged for 5 min at 1000 rpm. As the supernatant was removed, cells were resuspended in 1 × binding buffer (190 μL) with 10 μL PI. A CytoFLEX S flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) was adopted for cell apoptosis analysis.

Cell Transfection.
e overexpression vector (pcDN-A3.1/+vector; V79020, Invitrogen, ermo Fisher Scientific, USA) carrying NLRP3 gene was transfected into acinar cells using Lipofectamine 2000 Transfection Reagent (11668500, ermo Fisher Scientific, USA), with the empty vector used as a negative control (NC). Briefly, after being digested with trypsin (C0205, Beyotime Biotechnology, China), cells were seeded in a 24-well plate at 1 × 10 5 cells/well and incubated until 70-90% fusion. Lipofectamine 2000 (2.0 µL, at room temperature for 5 min) and DNA (0.8 µg) were diluted in 50 µL Opti-MEM I Reduced Serum Medium without serum (31985062, ermo Fisher Scientific, USA). en, the diluted DNA was mixed well with the dilute Lipofectamine 2000 and cultured for 20 min at room temperature, subsequent to which the 100 µL complexes were added into each well and mixed completely by a rocking plate back and forth. Cells were incubated for 48 h in a CO 2 incubator at 37°C.

Ruscogenin Ameliorated the SFR and Submandibular
Gland Inflammation of NOD/ShiLtJ Mice. Figure 1(a) shows the chemical structure of Ruscogenin. e SFR did not differ among each group in the early stage. From week 11 to 20, it was viewed that the SFR gradually reduced in NOD/ShiLtJ and vehicle groups (Figure 1(b)), whereas the SFR of mice treated with Ruscogenin did not decrease in comparison with mice treated with vehicle ( Figure 1(b), p < 0.05), which indicated that Ruscogenin could restore the SS-like symptom. rough histological analysis of submandibular gland, we discovered that both NOD/ShiLtJ and vehicle groups exhibited great infiltration of lymphocytes as well as more inflammation focuses with large area (Figure 1(c)). But the treatment of 0.3 mg/kg Ruscogenin decreased lymphocyte infiltration, the area, and a number of inflammation focuses, with 1 mg/kg Ruscogenin further enhancing that reduction (Figure 1(c)). Similarly, no significant difference in IL-6, IL-17, TNF-α, and IL-1β mRNA expression was presented in NOD/ShiLtJ and vehicle groups (Figure 1(d)), whereas IL-6, IL-17, TNF-α, and IL-1β mRNA expression in submandibular gland tissues prominently decreased in the Ruscogenin 0.3 group and the Ruscogenin 1 group when compared to the vehicle group, as the effect of 1 mg/kg Ruscogenin was better than 0.3 mg/kg Ruscogenin (Figure 1(d), p < 0.01). ose findings suggested that Ruscogenin improved the SFR and submandibular gland inflammation in a dose-dependent manner.

Ruscogenin Inhibited the Apoptosis of Acinar Cells in NOD/ShiLtJ Mice and Reversed TNF-α-Induced Apoptosis and
Inflammation of Acinar Cells. When compared to normal mice (BALB/c), apoptosis of acinar cells in NOD/ShiLtJ mice obviously elevated, whereas the treatment of 1 mg/kg Ruscogenin notably decreased the acinar cell apoptosis of NOD/ ShiLtJ mice (Figure 3(a)). To explore the function of Ruscogenin on TNF-α-induced acinar cell injury, the acinar cells were cultured with Ruscogenin at different doses (0.1, 1, and 10 µM) prior to exposure to TNF-α. e consequence of flow cytometry assay showed a marked rise in the apoptosis rate in cells treated with TNF-α (Figures 3(b) and 3(c), p < 0.001), while the treatment of Ruscogenin decreased cell apoptosis induced by TNF-α as the concentration of Ruscogenin increased (Figures 3(b) and 3(c), p < 0.01). Furthermore, cells in the TNF-α group exhibited lower AQP5 and AQP4 protein levels with higher NLRP3, caspase 1, and IL-1β expression than the control group (Figures 3(d)-3(g), p < 0.001). Nevertheless, with the dose of Ruscogenin elevated, cells with cotreatment of TNF-α and Ruscogenin gradually elevated AQP5 and AQP4 levels and reduced NLRP3, caspase 1, and IL-1β expression in comparison with cells treated with TNF-α alone (Figures 3(d)-3(g), p < 0.05).
ose data implied that Ruscogenin was able to protect acinar cells against TNF-α-induced injury.

Discussion
SS is an autoimmune disease predominantly affecting women, with major clinical characterizations comprising dry eyes and dry mouth [2,26]. Ruscogenin is an active component of Ophiopogon japonicas, which exerts various pharmacological properties including anti-inflammatory [23]. However, the functions of Ruscogenin on SS still remain unclear.
We firstly experimented in vivo through establishing animal models of SS with the treatment of Ruscogenin to explore the role of Ruscogenin in SS. In agreement with the research studies about the effect of MDG-1 and green tea polyphenols, as well as AT-RvD1 in SS [12,16,27], our study discovered that Ruscogenin recovered the SFR of mice and repressed lymphocytic infiltration and inflammation of submandibular glands, as higher dose of Ruscogenin exhibited better efficacy, which validated the protective role of Ruscogenin against SS. IL-6, IL-17, TNF-α, and IL-1β are crucial pro-inflammatory cytokines triggering intense inflammatory reaction, whose upregulation has been reported to associate with the pathogenesis of SS [28,29]. Former studies have shown several promising strategies that can attenuate SS via suppressing inflammation, and Ruscogenin has been proven to restrain inflammation in cerebral ischemic injury, acute lung injury, and blood-brain barrier dysfunction induced by cerebral ischemia [2,12,22,23,[29][30][31][32][33]. Similarly, during our experiments, we observed that Ruscogenin  Evidence-Based Complementary and Alternative Medicine prominently inhibited those inflammation-related factors in NOD/ShiLtJ mice with the increasing concentration of Ruscogenin, implying that Ruscogenin might restore SS through the inhibition of inflammation via the downregulation of pro-inflammatory cytokines in a dose-dependent manner.
Belonging to the AQP family, which is a group of specific water channels allowing the water to move in and out of cells to respond to osmotic/hydrostatic pressure gradients, AQP5 and AQP4 play a key part in forming tears and saliva. AQP5 is located in apical acinar and ductal cells in the lacrimal glands but apically at the membranes of acinar cells in salivary glands, which is believed to provoke the water outflow into the acinar lumen. As for AQP4, it is located laterally in acinar cells of the lacrimal glands, whereas in salivary glands, it is located basolateral membranes of acinar cells [34]. Previous reports have revealed a low expression as well as a disorder of AQP5 and AQP4 in SS animal models and SS patients. And the mutant of AQP5 is confirmed to be correlative with the decrease in the secretion of the salivary gland [2,25,34]. It has been reported that mesenchymal stem cells extract-based treatment could improve SS-associated symptoms through upregulation of AQP5 and AQP4 [2]. Consistent with the finding, we also found that Ruscogenin advanced AQP5 and AQP4 levels in NOD/ShiLtJ mice, implying the alleviating potential of Ruscogenin in SS through upregulation of AQP5 and AQP4.
As a vital element in inflammatory responses, the NLRP3 inflammasome, which is a multiprotein complex containing NLRP3 and caspase 1, participates in a variety of diseases including SS [11,35]. Besides, the involvement of NLRP3 inflammasome has also been confirmed in the effects of Ruscogenin on cerebral ischemia-induced dysfunction of blood-brain barrier [23]. us, it could be presumed that the protective role of Ruscogenin in SS might partially attribute to its regulation on NLRP3 inflammasome. To address that, western blot was performed after the treatment of Ruscogenin to evaluate protein levels of factors related to NLRP3 inflammasome. P2X7R is taken as a pivotal receptor in inflammation, whose activation positively connects with the maturation and release of pro-inflammatory molecules like IL-1β. In addition, P2X7R is verified to cause inflammatory reactions and exacerbate the prognosis through strongly activating NLRP3 inflammasome [36][37][38][39]. Similar to the study about Ruscogenin in blood-brain barrier dysfunction [23], our research viewed that Ruscogenin repressed P2X7R, NLRP3, caspase 1, and IL-1β expression in SS mice models in a dose-dependent manner, which implicated that the protective effect of Ruscogenin against SS might be associated with NLRP3 inflammasome.
Secondly, acinar cells were isolated to further investigate the role of Ruscogenin in cells. rough the AO/PI staining assay, it was discovered that Ruscogenin suppressed the elevating apoptosis of acinar cells in NOD/ShiLtJ mice, revealing that perhaps Ruscogenin promoted the preservation of acinar cells. In order to validate whether Ruscogenin could protect acinar cells from inflammation, cells were treated with TNF-α to construct a model of cell injury due to the fact that TNF-α is capable of inducing cytotoxicity in various kinds of cells [27]. e result of the flow cytometry assay and Western blot presented that TNF-α advanced cell apoptosis and pro-inflammatory cytokines while inhibiting AQPs. e similar consequence has been acquired in previous works about the effect of TNF-α [13,25,[40][41][42]. ose data showed a successful establishment of TNF-α-induced cell injury model. MDG-1 has been proved to repress cell apoptosis and inflammation induced by H2O2 [31]  Representative images of cell apoptosis (b) and apoptosis rates (c) were evaluated through flow cytometry assay after the treatment of TNF-α and Ruscogenin. (d and e) Representative images of protein bands (d) and relative protein expression of AQP5 and AQP4 (e) in acinar cells was tested by western blot after the treatment of TNF-α and Ruscogenin. GAPDH is a loading control. (f and g) Representative images of protein bands (f ) and relative protein expression of NLRP3, caspase 1, and IL-1β (g) in acinar cells was assessed by western blot after the treatment of TNF-α and Ruscogenin. GAPDH is a loading control. * * * p < 0.001 vs. control group; ∧ p < 0.05, ∧∧ p < 0.01, ∧∧∧ p < 0.001 vs. TNF-α group. All experiments were repeated independently at least three times. Data were performed as the means ± standard deviation. TNF: tumor necrosis factor; AO/PI: acridine orange and propidium iodide; AQP: aquaporin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; NLRP3: nucleotide binding oligomerization domain-like receptor 3; IL: interleukin. 8 Evidence-Based Complementary and Alternative Medicine Ruscogenin reversed those functions of TNF-α above in a dose-dependent manner, implying that Ruscogenin indeed protected acinar cells from cell injury via the inhibition of inflammation.
To probe into the specific mechanism of Ruscogenin against SS, NLRP3 overexpression plasmid was transfected into acinar cells, with western blot testing transfection efficiency. Consequently, we found the inhibitory effect of Ruscogenin on TNF-α-induced upregulation of NLRP3 inflammasome-related as well as pro-apoptotic (Bax [43]) molecules and downregulation of anti-apoptotic factor (Bcl-2 [43]) were reversed by NLRP3 overexpression plasmid, which suggested that Ruscogenin defended against inflammation and apoptosis in acinar cells via negatively Representative images of protein bands (a) and relative protein expression of NLRP3 in acinar cells was tested by western blot after the transfection of NLRP3 overexpression plasmid. GAPDH is a loading control. (c and d) Representative images of protein bands (c) and relative protein expression of NLRP3, caspase 1, and IL-1β (d) in acinar cells was assessed by western blot after the treatment of TNF-α and Ruscogenin as well as transfection of NLRP3 overexpression plasmid. GAPDH is a loading control. (e and f ) Representative images of protein bands (e) and relative protein expression of Bax and Bcl-2 (f ) in acinar cells was assessed by western blot after the treatment of TNFα and Ruscogenin as well as transfection of NLRP3 overexpression plasmid. GAPDH is a loading control. † † † p < 0.001 vs. NC group; * * * p < 0.001 vs. Control group; ∧∧∧ p < 0.001 vs. TNF-α group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. TNF-α+Ruscogenin 10 group. All experiments were repeated independently at least three times. Data were performed as the means ± standard deviation. NLRP3: nucleotide binding oligomerization domain-like receptor 3; TNF: tumor necrosis factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IL: interleukin; NC: negative control for NLRP3 overexpression plasmid. mediating NLRP3 inflammasome. Moreover, in an in vivo experiment, we found that the ameliorate effect of 1 mg/kg Ruscogenin on the NOD/ShiLtJ mice was better than that of 0.3 mg/kg Ruscogenin; in an in vitro experiment, the effect of 10 µM Ruscogenin on the TNF-α-induced acinar cells was better than that of 0.1 and 1 µM Ruscogenin.

Conclusion
As a conclusion, the research clarified a positive role of Ruscogenin against SS and demonstrated that Ruscogenin ameliorated SS by inhibiting NLRP3 inflammasome activation through experiments in vivo and in vitro, providing an innovative and potential drug for SS treatment. But more studies and clinical trials are necessary for further determination of the Ruscogenin's efficacy on SS. In the future research, we will continue to emphasize on more targets of Ruscogenin as well as their mechanisms of action to improve SS management.
Data Availability e analyzed datasets generated during the study are available from the corresponding author on reasonable request.

Ethical Approval
All animal experiments were performed in accordance with the guidelines of the China Council on Animal Care and Use.
is study was approved by the Committee of Experimental Animals of Nanfang Hospital (NF20200815010). Every effort was made to minimize pain and discomfort to the animals. e experiments on animals were performed in Nanfang Hospital.

Conflicts of Interest
e authors declare no conflicts of interest.