Inhibition of Endoplasmic Reticulum Stress by 4-Phenyl Butyric Acid Presents Therapeutic Effects on Periodontitis: Experimental Studies In Vitro and in Rats

This study investigated the probable mechanisms of endoplasmic reticulum (ER) stress involved in periodontitis in vitro and in vivo. We isolated periodontal ligament stem cells from periodontitis patients and healthy controls (P-PDLSCs and H-PDLSCs). To further simulate the periodontal microenvironment in patients, lipopolysaccharide (LPS) was used to treat H-PDLSCs. The results showed that periodontitis-related inflammation gave rise to the upregulated expression levels of ER stress representative genes including GRP78, PERK, ATF4, and CHOP. In contrast, the treatment of 4-phenyl butyric acid (4-PBA) remarkably suppressed ER stress and supported cell viability. The increased secretion of proinflammatory factors like TNF-α, IL-1β, and IL-6 and the activation of NF-κB pathway were also attenuated by 4-PBA treatment. Moreover, 4-PBA treatment restored the impaired osteogenic differentiation ability of PDLSCs, as demonstrated by the upregulated expression levels of Runx2 and OCN as well as the enhanced Alizarin red staining. Local administration of 4-PBA could rescue alveolar bone resorption of LPS-induced periodontitis rats. Thus, our findings suggested ER stress might act as a promising therapeutic target against periodontitis.


Introduction
Periodontitis is among world's most prevalent inflammatory diseases, which has destructive effects on periodontal tissues including gingival, cementum, periodontal ligament, and alveolar bone [1]. It is reported that three quarters of the adult population worldwide have mild periodontal disease (gingivitis) at least and more than one-fifth present with severe and destructive periodontitis [2]. The clinical symp-toms include gingival bleeding, formation of periodontal pocket, loss of connective tissue attachment, alveolar bone resorption, and eventually teeth exfoliation. The pathogenesis of periodontitis involves a local inflammatory reaction triggered by the presence of bacterial plaque [3]. Thus, the ultimate objective of periodontal treatment is not simply to control the inflammation, but more importantly, to recover the structure and feature of the damaged tissues in a diseased microenvironment.
Periodontal ligament stem cells (PDLSCs) are a group of heterogeneous mesenchymal stem cells that are located in the periodontal ligament [4]. It is widely accepted that the augmented aberrant differentiation and osteogenic differentiation dysfunction of PDLSCs are closely associated with the alveolar bone resorption during periodontitis [5,6]. A complex network of signaling pathways governs the differentiation of PDLSCs in periodontitis. Generally, signaling pathways related to cell injury all are considered to be associated to the differentiation of PDLSCs, such as inflammation, autophagy-lysosome pathway, calcium homeostasis, ubiquitin-proteasome system, and endoplasmic reticulum (ER) stress. Generally, the inflammatory microenvironment composed of inflammatory cells and cytokines is noticeable to impede the osteogenic differentiation ability of PDLSCs. Nevertheless, anti-inflammatory therapy still has not achieved the goal to reserve aberrant differentiation of PDLSCs completely, implying there may be other regulatory mechanisms in periodontitis [7,8]. Our previous studies demonstrated that unfolded protein accumulation in the ER activates the canonical unfolded protein response (UPR) representative genes including GRP78, PERK, ATF4, and CHOP and subsequently results in ER stress response, which may facilitate the aggravation of periodontitis [7,9,10]. Actually, numerous evidences have documented the intersections between ER stress and inflammation. For example, IREα, a typical transmembrane receptor involved in UPR, could recruit TRAF2 to the ER membrane via the NF-κB pathway to initiate cellular inflammatory responses [11]. However, there is still no available information to provide insight into a communicating network between ER stress and PDLSC fate determination especially osteogenic differentiation in periodontitis.
In the present study, we investigated the probable mechanisms of ER stress in periodontitis in vitro and in vivo. We found that 4-phenyl butyric acid (4-PBA) confers therapeutic effects on periodontitis by suppressing ER stress and inflammation and restoring PDLSC function. Our findings suggested that targeting ER stress might provide a prospective therapeutic strategy for periodontitis.

2.2.
Real-Time Quantitative PCR. By using the SYBR Premix Ex Taq II kit (Takara, Shiga, Japan), RT-qPCR reactions were performed by using CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). We used standard settings: 94°C, 3 min; 94°C, 15 s; 60°C, 30 s; repeated 40 cycles, and then dissociation. Each assay ran in triplicate. The relative standard curve method calculated the arbitrary mRNA concentrations. The ΔΔ -ct method normalized to GAPDH. The primer sequences used in the present study are showed in Table 1. 2.3. Western Blot Analysis. Western blot analysis was performed as described in our previous studies [7,9,10]. RIPA lysis buffer extracted proteins, and then, G250 protein assay (Beyotime, Jiangsu, China) extracted soluble protein. Protein samples were loaded on 10% sodium dodecyl sulfate polyacrylamide gels (Invitrogen), transferred to polyvinylidene fluoride membranes (Bio-Rad), and blocked with 5% nonfat milk powder. Membranes were incubated overnight with the following primary antibodies for human Runx2 (Cell Signaling, Beverly, MA, USA), OCN (Santa Cruz, Dallas, TX, USA), NF-κB (Santa Cruz), p-NF-κB (Santa Cruz), and βactin (Cowin Biotech, Beijing, China). Torseradish peroxidase-(HRP-) conjugated secondary antibody (Cowin Biotech) incubated the membranes for 2 h at room temperature. Protein signals were visualized by using the ECL Western Blotting Detection System (GE Healthcare, Piscataway, NJ, USA).
2.4. Enzyme-Linked Immunosorbent Assay (ELISA). The samples were centrifuged at for 5 min 3000 g after a 15 min elution phase at room temperature in Eppendorf tube with 50 ml of PBS (Sigma-Aldrich). The supernatants were stored at -80°C until used. The concentrations of proinflammatory

CCK-8 Assay.
A total of 1 × 10 3 cells of 0.1 ml per well were cultured in a 96-well plate in 10% FBS in three replicate wells. After drug treatment, 10 μl of CCK-8 kit reagent (Beyotime) was added to 100 μl of cell culture medium and incubated for 1.5 h at 37°C. The absorbance of each well at 450 nm was measured by using a microplate reader (Bio-Rad).
2.6. Osteogenic Differentiation In Vitro. The medium was changed to 2 mol/l β-glycerophosphate, 10 nmol/l dexamethasone, and 100 μg/ml ascorbic acid (Sigma-Aldrich). RNA was extracted for 1 week for the osteogenic genes by using RT-qPCR analysis. Protein was extracted after 14 days for osteogenic markers by using Western blot analysis. Osteoblast calcium deposits were determined by staining with 1% Alizarin Red (Sigma-Aldrich) as previously described [14]. 1% cetylpyridinium chloride (Sigma-Aldrich) dissolved the mineralized nodules, and a spectrophotometer (Epoch, Bio-Tek, USA) measured the OD value at 562 nm. Total protein quantity of each sample normalized the results to exclude the impact of cell numbers.

Drug Administration in Sprague-Dawley (SD) Rats of Experimental Periodontitis.
A periodontitis model was established as described before [15]. We distributed three groups of nine eight-week-old SD rats: saline; LPS (10 μg/day) and LPS (10 μg/day) +4-PBA (5 nmol/day) with three rats per group. Then, the drug was injected between the first and second upper molars into the maxillary palatal gingiva in each group and repeated every two days for 7 days. The rats were anesthetized and euthanized by exsanguination. The SD rats' whole heads were removed, and a micro-CT system (Siemens Inveon Micro-CT, Munich, Germany) scanned and analyzed the maxillaries. The alveolar bone height of four different sites was recorded by measuring the length from the alveolar bone crest to the cemento-enamel junction (CEJ) in two molars. Body weight, heart/body weight, liver/body weight, spleen/body weight, adrenal gland/body weight, and kidney/body weight were measured to appraise the effect of drug administration on general condition.

Statistical
Analysis. Data are represented as means ± standard deviations of each independent experiment (n = 3 ). Comparisons between the two groups were calculated by independent two-tailed unpaired Student's t-test and among numerous comparisons by the Bonferroni correction of analysis of variance in the GraphPad Prism software (San Diego, CA, USA). Significant differences were considered when P < 0 05.

4-PBA Reverses ER Stress in PDLSCs under Inflammatory
Periodontitis Condition. Our previous study has identified the upregulation of the classical UPR genes including GRP78, PERK, ATF4, and CHOP in primary P-PDLSCs [7].
To testify the effect of 4-PBA on ER stress in the inflammatory microenvironment, we treated P-PDLSCs with 4-PBA (5 mM) for 7 days. As shown in Figure 1(a), the UPR target genes in P-PDLSCs were significantly downregulated. To further simulate the periodontal microenvironment in patients, LPS was used to treat H-PDLSCs. The consequences showed that 4-PBA could significantly decrease the levels of URP target genes in LPS-treated H-PDLSCs. Nevertheless, their expression levels were still expressively higher than those in H-PDLSCs (Figure 1(b)).   Figures 2(a) and 2(b)). To explore the underlying mechanism, Western blot analysis was used to determine the expressions of p-NF-κB and NF-κB, and the results showed that treatment of 4-PBA could remarkably reduce

4-PBA Restores the Impaired Osteogenic Differentiation
Ability of PDLSCs in the Inflammatory Microenvironment. As shown in Figure 3, RT-qPCR (Figures 3(a) and 3(b)) and Western blot results (Figures 3(c)-3(f)) showed that the osteogenic differentiation associated biomarker proteins, like Runx2 and OCN, was significantly upregulated after 4-PBA treatment in both P-PDLSCs and LPS-treated H-PDLSCs. In addition, Alizarin red staining demonstrated that 4-PBA-treated cells formed more mineralization nodules in PDLSCs in the inflammatory microenvironment (Figures 4(a) and 4(b)).

Local Administration of 4-PBA Rescues Alveolar Bone
Resorption in an LPS-Induced Periodontitis Rat Model. To further examine the effect of 4-PBA on periodontitis in vivo, LPS administration was recruited to establish a periodontitis rat model. As shown in Figure 5, the LPS group showed more excessive bone resorption as the farthest dis-tance, from the alveolar bone crest to the CEJ, was observed in all the six sites. Whereas in the LPS+4-PBA group, the alveolar bone resorption of three molars was notably lessened, indicating that 4-PBA could rescue alveolar bone resorption in periodontitis. On the other hand, drug administration including LPS and LPS+4-PBA had little effect on the general condition of control rats ( Table 2). Body weight, heart/body weight, spleen/body weight, adrenal gland/body weight, and kidney/body weight had little difference between the three groups, while liver/body weight was decreased in the LPS group compared to the control group and the LPS+4-PBA group ( Table 2).

Discussion
In the present study, we provided a communicating network between ER stress and PDLSC function in periodontitisrelated inflammation. To this end, P-PDLSCs and H-PDLSCs used as model cells and 4-PBA, an identified ER stress inhibitor, were used in this study. To simulate inflammatory periodontitis condition in vitro and in vivo, LPS was used to treat H-PDLSCs and also locally administrated in rats. The results showed that 4-PBA could downregulate  Due to their self-renewal potential and multidifferentiation capability, PDLSCs have been widely used in tissue regeneration including periodontium [16]. The function and fate of PDLSCs are closely associated with the pathogenesis, progression, treatment, and prognosis in periodontitis [17,18]. However, PDLSCs from periodontitis patients show deficient osteogenic differentiation ability in comparison with cells from healthy individuals [19]. Interestingly, osteogenic differentiation deficiency could not be retrieved after ex vivo culture and expansion [20]. In addition to chronic inflammation, the osteogenic capacity of PDLSCs might be affected by other factors. ER stress, caused by UPR, is an essential strategy in response to the drastic changes of the extracellular environment, which has been demonstrated to be involved in multiple oral diseases [7,21,22]. As previous studies reported, the IRE1α branch of the UPR might be associated with the secretion of proinflammatory cytokines in many kinds of cells such as endothelial cells and macrophages [23]. Endoplasmic reticulum stress impels macrophages to induce mature IL-1β in response to TLR4 stimulation via a TRIF-and caspase-8-dependent pathway [24]. In the present study, we found the upregulated expression levels of ER stress representative genes GRP78, PERK, ATF4, and CHOP both in P-PDLSCs and LPS-treated H-PDLSCs. These results indicated ER stress is a crucial pathogenesis in periodontitis. Moreover, periodontitis-related inflammation in the microenvironment resulted in lower cell viability, secretion of proinflammatory factors, and activation of NF-κB pathway. In accordance with our previous studies [7,21,22], periodontitis impaired ER function and causes ER stress, which in turn results in deficient osteogenic differentiation of PDLSCs.
To investigate functional significance of ER stress in periodontitis, an identified ER stress inhibitor 4-PBA was used here. Previous studies have showed the therapeutic potential of 4-PBA for many kinds of human diseases [25][26][27]. Mechanically, 4-PBA could repress the overactivated ER stress and attenuate the UPR by stabilizing protein conformation, enhancing folding capacity of ER, and facilitating trafficking of mutant proteins [28][29][30][31]. In the present study, we found that the 4-PBA treatment remarkably suppressed ER stress and supported cell viability. The treatment of 4-PBA also reduced the enhanced secretion of proinflammatory cytokines by suppressing the activation of NF-κB pathway. Indeed, previous studies have demonstrated that ER stress has been related to the activation of NF-κB, a crucial transcription factor for many inflammatory processes [32]. We demonstrated that 4-PBA could interfere with the inflammation-related pathways in periodontitis, which might provide a new insight into the therapeutic mechanism of 4-PBA. Furthermore, 4-PBA treatment restored the impaired osteogenic differentiation ability of PDLSCs in the inflammatory microenvironment, and local administration of 4-PBA could rescue alveolar bone resorption of LPSinduced periodontitis rats. Our previous studies have shown that ER stress negatively affects the osteogenic differentiation function of PDLSCs via PERK and IRE1α pathways [7,8]. Indeed, accumulating experimental evidence suggested the harnessing of ER stress has proven to be a particularly plausible therapeutic strategy for regulating bone metabolism and tissue repair, such as osteoarthritis [33], intervertebral disc degeneration [34], osteogenesis imperfecta [35,36], and implant osseointegration [37]. It is noted that drug administration of LPS or LPS+4-PBA had adverse effects on the general condition of rats with lower liver/body weight than that of the control group, which may result from the hepatotoxicity of LPS [38].
Although our findings demonstrated that 4-PBA could suppress inflammation and restore the impaired osteogenic differentiation ability of PDLSCs by affecting the NF-κB pathway, other molecular mechanisms have not been further studied. We also did not use multiple models of periodontitis, such as bacteria, ligature, or hypoxia, to validate these findings. These deficiencies will be explored in depth in future studies.

Conclusion
The present study investigated the possible mechanism ER stress associated with periodontitis. We investigated that inhibition of endoplasmic reticulum stress by 4-PBA suppressed ER stress and inflammation, supported cell viability, and restored the defective osteogenic differentiation of PDLSCs under inflammatory periodontitis conditions. Local administration of 4-PBA rescued alveolar bone resorption in an LPS-induced periodontitis rat model. Our findings suggested the potential of harnessing ER stress in order to develop a novel therapeutic approach for periodontitis.

Data Availability
All data generated or analyzed during this study are included in this published article.

Conflicts of Interest
The authors declare that no competing interests exist.