Protective Effects of Collagen Peptide on Periodontal Ligament Fibroblasts

To study the protective effect of collagen peptide on human periodontal ligament fibroblasts (hPDLFs) induced by lipopolysaccharide and its possible mechanism. Lipopoly-saccharide was used as a pathogenic factor to stimulate hPDLFs. Then, interleukin-8 and tumor necrosis factor-α in the supernatant were quantified, and Toll-like receptor 4 expression and the activation of nuclear factor-κB pathway were also studied. The results showed that collagen peptide above 100 mg/L could effectively reduce the toxicity of lipopolysaccharide, and significantly reduce interleukin-8 and tumor necrosis factor-α produced by lipopolysaccharide. Suggesting that collagen peptide can provide effecitve and safe solution to oral cavity repair.


Introduction
Periodontitis is a prevalent disease in stomatology, with a high rate of morbidity.Its clinical features include inflammatory destruction of the gingiva, periodontal ligament, cementum, and alveolar bone [1].Periodontal ligament fibroblasts (PDLFs), the most widely distributed cells with a critical function in the periodontal ligament [2], have long been the focus of pathogenesis research, preventive nursing, and restorative treatment of periodontitis.Regulation of PDLF apoptosis and the secretion of inflammatory factors is critical to the prevention and treatment of periodontitis.Gram-negative anaerobic bacilli are the major pathogens in periodontitis; their outer membrane component, lipopolysaccharide (LPS), is considered to be one of the main triggers for the destruction of periodontal tissues [3].During the development of periodontitis, LPS can enter the deep periodontal tissue through damaged epithelium and directly act on PDLFs, triggering and exacerbating inflammatory responses in periodontal tissues [4].
Collagen peptide (CP) is the hydrolysate of collagen; it can resist oxidation and bacteria, regulate immunity, and promote wound healing [5].As a natural biological product, CP has been exploited in food, cosmetics, and medicine, but few studies have examined its use in the repair of the oral cavity.In this study, we tested how CP influences PDLF cell function, including its immunomodulatory effects.In short, normal hPDLFs were cultured and stimulated with LPS in vitro to simulate an inflammatory response.Then, following treatment with different concentrations of CP, we quantified interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) in the supernatant, the LPS target Toll-like receptor 4 (TLR4) expression, and the activation of nuclear factor-κB (NF-κB) pathway.Our findings provide a foundation for the potential clinical use of CP in the prevention and treatment of periodontitis.

Chemicals, Reagents and Kits
Collagen peptide supplied by Beijing Semnl Biotechnology Co., Ltd.(Beijing, China).hPDLFs were purchased from American Type Culture Collection.Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO Inc. (NY, USA).Streptomycin and penicillin were purchased from Sigma.IL-8 and TNF-α ELISA kits were purchased from Sino Biological Inc. (Beijing, China).TLR4, NF-κB p6, and GAPDH antibodies were purchased from Abcam (Cambridge, MA,USA).

Grouping
Because the CCK-8 (cell viability by cell counting kit-8) assay indicated that 10 mg/L LPS strongly inhibited the growth of hPDLFs without obvious cytotoxicity, this dose was used in all subsequent experiments.
HPDLFs were divided into 6 groups: blank control group, model group, CP-10 group (10 mg/L CP), CP-50 group (50 mg/L CP), CP-100 group (100 mg/L CP), CP-500 group (500 mg/L CP), and CP-1000 group (1000 mg/L CP).DMEM was used for the blank control group, DMEM + 10 mg/L LPS in the model group, and DMEM + 10 mg/L LPS + CP at the corresponding doses were used in the other groups.

Detection of CCK-8 Assay
Cells in the logarithmic growth phase were digested into a cell suspension (1×10 5 cells/mL), and distributed into a 96-well plate (100 μL/well), 100 μL of PBS was added to the surrounding wells.The cells were then cultured for 24 h, the medium was discarded, and DMEM containing the corresponding analyte was added to each group.After 24 h, 10 μL of CCK-8 (Dojindo Laboratories Kumamoto, Japan) was added to each well and mixed well by gently tapping the culture plate, then incubated in the dark for 2 h.Finally, the mixture was gently shaken for 10 min and the absorbance (A) value of each well was measured at 450 nm using a microplate reader.
Relative proliferation rate:

Wound Healing Assay
Cells in the logarithmic growth phase were digested into a cell suspension (1×10 5 cells/mL), and distributed into a 6-well plate.At 100% confluence, the cell layer was scratched with a 200 μL sterile pipette tip in a straight line, and the shed cells were removed by two gentle PBS washes.Then, DMEM containing the corresponding analyte was added to each group, and the cells were observed and imaged under a microscope (DMIL LED, Leica, Wetzlar, Germany) at 0, 24, and 48 h after the scratch.The wound healing rate was calculated to assess the cell migration ability.

Enzyme-linked immunosorbent assay (ELISA) cytokine quantification
Cells in the logarithmic growth phase were digested into a cell suspension (1×10 5 cells/mL), and distributed into a 6-well plate.DMEM containing the corresponding analyte was added to each group.
After 24 h, cell supernatant was collected, and the IL-8 and TNF-α levels per mg protein were quantified using ELISA kit based on the manufacturer's protocol.

Detection of Protein Expression by Western Blotting
Cells in the logarithmic growth phase were digested into a cell suspension (1×10 5 cells/mL), and distributed into a 6-well plate.DMEM containing the corresponding analyte was added to each group.After 24 h, the cells were collected, the total protein was extracted and quantified with a BCA assay.30 μg of protein from each sample was subjected to electrophoresis, transferred to a membrane, blocked with skim milk powder, and incubated with primary antibodies against TLR4, NF-κB p6, and GAPDH at 4 ℃ overnight.After washing with TBST, the membrane was incubated with secondary antibodies at room temperature for 1 h, bathed in ECL substrate, and the protein bands were observed.

Effect of CP on hPDLF Viability
As shown in Figure 1, CP promoted the viability of hPDLFs at 1-5 d, and its effect was enhanced with time.Among the different doses, 100 mg/L had the largest impact on viability.

Effect of CP on the Viability of LPS-treated hPDLFs
As shown in Figure 2, the model group cell viability was significantly lower than in the blank control group, indicating that 10 mg/L LPS reduced hPDLF cell viability.CP at a concentration >50 mg/L reduced LPS cytotoxic effects on hPDLFs, greatly increasing the viability of hPDLFs.The CP-10 group had similar viability to the model group, while the cell viability in CP-50, CP-100, CP-500, and CP-1000 groups was significantly higher than in the model group.However, cell viability was not significantly different between CP-100, CP-500, and CP-1000 groups, suggesting an all-or-nothing effect that is not increased with increasing concentrations.

Effect of CP on Cytokine Secretion Following LPS Treatment
As shown in Figure 3, the blank control group showed a high migration ability; the scratch distance was significantly reduced by 24 h and almost recovered at 48 h.Cell migration was impaired in the model group.Migration was similarly poor in the CP-10 group, but significantly higher in the CP-50, CP-100, CP-500, and CP-1000 groups, compared to the model group.At 24 h, the cell migration abilities of the CP-50, CP-100, CP-500, and CP-1000 groups were no different than the blank control group.At 48 h, cell migration ability was similar between the CP-100 and blank control groups.As such, LPS inhibits the migration of hPDLFs and CP can attenuate this effect, enhancing the migration ability of hPDLFs.

Effect of CP on LPS-treated hPDLF Proteins
As shown in Figure 4, the pro-inflammatory chemokine IL-8 was significantly increased in hPDLFs after LPS stimulation for 24 h.Compared to the blank control group, IL-8 was around 5 times higher, TNF-α was around 4 times higher.This indicates that LPS can significantly increase IL-8 and TNF-α secretion in hPDLFs.Compared to the model group, the levels of IL-8 and TNF-α were similar in CP-10 but in the CP-50, CP-100, CP-500, and CP-1000 groups IL-8 and TNF-α were decreased.These findings demonstrate that CP at a concentration >50 mg/L can decrease IL-8 and TNF-α, thus significantly attenuatingy the effect of LPS on hPDLFs.

Effect of CP LPS-treated hPDLF Migration
As shown in Figure 5, compared with the blank control group, the model group had significantly increased TLR4 and NF-κB p65.TLR4 and NF-κB p65 were similar in CP-10, CP-50, and the model group.Levels of these proteins were similar b etween CP-100, CP-500, CP-1000, and the blank control group.These results demonstrate that LPS increases TLR4 and NF-κB p65 in hPDLFs.CP at a concentration >100 mg/L can attenuate these LPS effects.

Discussion
As the most important cells in the periodontal ligament, PDLFs can synthesize collagen, elastic fibers, and glycoproteins [7].They can absorb collagen and phagocytose foreign bodies, and possess many biological functions such as chemotaxis, adhesion, proliferation, and differentiation into cementoblasts and osteoblasts.As such, they are critical for periodontal tissue regeneration and injury repair, and are the main source of cells for new attachments to the gingiva and root surfaces following periodontal treatment [8].Therefore, protecting the normal viability and migration ability of PDLFs is important for preventing the occurrence and development of periodontitis, and in the repair and reconstruction of periodontal tissues.In this study, we found that CP at a concentration >100 mg/L facilitated the growth of hPDLFs, reduced the cytotoxic effects of LPS, and enhanced the viability and migration of hPDLFs.These findings are consistent with previously published work by Liu C et al [9].These data suggest that CP can facilitate oral cavity repair.PDLFs can synthesize, degrade and absorb collagen, and serve as the main source of collagen fibers in the periodontal ligament extracellular matrix.CP is the hydrolysis product of collagen and can contribute to the growth of fibroblasts and the synthesis of collagen [10].It was, therefore, speculated that CP can act as a signaling molecule to accelerate metabolic activity of PDLFs, and activate the collagen synthesis-related metabolic pathway in PDLFs, thereby facilitating cell viability and migration.

Figure 5. The Expression of hPDLFs Related Proteins in Different Groups Using Western Blotting
With an improved understanding of the pathogenesis of periodontitis, there has been a growing recognition of the importance of the host-pathogen response in triggering and worsening tissue damage [11].As such, strategies to reduce the expression of inflammatory factors and modulate the inflammatory response in periodontal tissues should play a role in the prevention and treatment of periodontitis.It has been shown that PDLFs have innate immune cell-like functions [12]; they recognize pathogen-associated molecular patterns and, in response, secrete cytokines and chemokines, thereby regulating the proliferation, migration, and activation of innate and adaptive immune cells.LPS is an important immunogenic factos on the surface of gram-negative bacteria; it triggers a wide range of biological activities, including the secretion of cytokines such as IL-8 and TNF-α by various cells [13].In this study, hPDLFs were stimulated with LPS to establish the in vitro periodontal infection model.The concentrations of inflammatory factors in the supernatant at 24 h were detected by ELISA, and 10 mg/L LPS increased the secretion of IL-8 and TNF-α in hPDLFs.
TNF-α, an inflammatory cytokine closely related to the osteogenic differentiation of stem cells, can promote the secretion of IL-1, IL-6, and IL-8; TNF-α antagonists can greatly reduce bone resorption in the animal periodontitis model [14].In this study, TNF-α secretion was higher in the model group compared to the control group; CP attenuated this increase, suggesting that CP can relieve the inflammatory response by reducing TNF-α.IL-8 is an important pro-inflammatory chemokine and immunoreg-ulatory factor in periodontitis, that can directly or indirectly promote inflammation resulting in alveolar bone resorption, collagenase production, inflammatory cell aggregation, etc. [15], thus leading to periodontal tissue destruction and facilitating the development of periodontal disease.The expression of IL-8 is cell, temperature, and LPS-dose-dependent.We found that CP at >100 mg/L significantly attenuated LPS-induced IL-8 secretion from hPDLFs, indicating that CP has a similar effect on IL-8 and TNF-α.
As a component of the gram-negative bacterial cell wall, LPS is characterized by high toxicity and antigenicity, and plays a key role in the occurrence and development of periodontal disease.TLR4 is an important receptor for LPS.By binding to TLR4, LPS initiates a cell signaling cascade that induces gene transcription, and leads to the release of a variety of cytokines, resulting in toxicity [16].TLR4, as a regulatory factor, can activate the transcription factor NF-κB, which then initiates the expression of inflammatory cascade effectors (IL-8 and TNF-α), inducing immuno-inflammatory responses.These effectors in turn induce the further activation of NF-κB, forming a positive feedback loop and worsening the inflammatory response.NF-κB is an important signaling pathway in the inflammatory response, which can regulate the transcription and expression of such inflammatory factors as TNF-α and IL-8 [17].In our study, we found that, as expected, LPS increased TLR4 and NF-κB p56 in hPDLFs; CP at >100 mg/L attenuated this, suggesting that CP is protective against LPS-induced inflammation in hPDLFs.The possible mechanism is that CP inhibits the secretion of the inflammatory cytokines TNF-α and IL-8 by regulating the TLR4/NF-κB pathway, thereby protecting hPDLFs.Zhang Y et al. [18] argued that CP can reduce the phosphorylation and nuclear translocation of NF-κB p65 in HaCaT cells, suggesting that the anti-inflammatory effect of CP on hPDLFs is also achieved by inhibiting the NF-κB pathway and its downstream factors such as TNF-α.
Temperature, humidity, and food residues in the oral cavity create a permissive environment for microbial growth, making the oral cavity one of the main channels for bacterial invasion into the human body.The self-purification function of the oral cavity is impaired during immunosuppression, allowing microbes to rapidly reproduce, resulting in periodontitis.Effective oral nursing can prevent or reduce the occurrence of periodontitis and other oral inflammation, but is reliant on drug therapy.Drugs can effectively control inflammation, but their long-term use is associated with serious adverse reactions, such as gastrointestinal, cardiovascular, hepatic, and renal problems.Therefore, antiinflammatory compounds with low toxicity are urgently needed.In this study, we demonstrated that CP can attenuate the LPS-induced hPDLF impaired immunomodulatory function, providing promising evidence to support the use of CP in the prevention and treatment of periodontal disease.CP is a natural active ingredient characterized by safety, stability, and broad effects.It has good biocompatibility, does not influence cell proliferation even after large doses, and can be added to food products.In addition, CP can also offer a more durable and safer solution to oral cavity repair.

Figure 1 .
Figure 1.Effect of Different Concentrations of CP on the Growth of Normal hPDLFs.

Figure 3 .
Figure 3. Cell Migration Ability of Different Groups of hPDLFs.

Figure 4 .
Figure 4. Detection of Inflammatory Factors Secreted by hPDLFs in Different Groups by ELISA.