Elevated Levels of Protein Disulfide Isomerase and Binding Immunoglobulin Protein Implicated in Spinal Cord Injury Paraplegia Patients with Pressure Ulcers

Abstract

Aims: To explore the associations between two endoplasmic reticulum (ER) stress proteins, protein disulfide isomerase (PDI), binding immunoglobulin protein (BIP), and the development and progression of pressure ulcers (PUs) in spinal cord injury (SCI) paraplegia patients. Methods: ELISA kits were used to measure the levels of serum PDI and BIP in 67 SCI paraplegia patients with PUs and 61 SCI paraplegia patients without PUs. The associations between PDI and BIP, PU formation, PU staging, and pressure ulcer scale for healing (PUSH) score were analyzed. Results: The patients in the PU group had higher levels of PDI and BIP than those in the non-PU group (both p < 0.05). Furthermore, the levels of PDI were positively correlated with those of BIP (r = 0.707, p < 0.0001). There were significant differences in the PDI and BIP levels among the different stages of PU (all p < 0.05). As the PU stages progressed, the levels of PDI and BIP first increased, then decreased, and finally peaked at stage III of the PUs. The PUSH scores significantly declined 7 days after debridement for the PU stage II (p < 0.01) but showed no significant difference between stages III and IV at 7 days after debridement (p > 0.05). The PUSH scores also decreased at 28 days after debridement for stages II, III, and IV (all p < 0.01). Higher PUSH scores indicated a longer time of debridement accompanied by a longer wound surface healing time (p < 0.05). Conclusion: ER stress proteins may be involved in the process of PU formation and healing; moreover, the levels of PDI and BIP were also associated with the severity of the PUs. Finally, we found that the PUSH scores can be used as a reference to evaluate PU severity and healing.

Introduction

Spinal cord injury (SCI) is a devastating condition causing significant personal and social burdens. It is accompanied by temporary or permanent changes or loss in the normal motor, sensory, or autonomic functions distal to the level of the trauma (Mothe and Tator, 2012). Pressure ulcers (PUs), also known as decubitus ulcers, are commonly considered to be a serious complication of SCIs, with an estimated 95% of individuals with SCIs developing PUs over the course of their lifetime (Nussbaum et al., 2013). PUs are soft tissue lesions that usually occur over bony prominences, including the sacrum, ankles, hips, coccyx, and heels, as a result of unrelieved pressure from perpendicular compression or tangential force (Hitzig et al., 2013). People with SCIs have a number of contributing or confounding factors, which are associated with a lifelong increased risk of PU formation, including impaired skin sensation, paralysis, reduced mobility, poor nutrition, anemia, spasticity, and skin maceration related to incontinence (Recio et al., 2012). Electrical stimulation is an advantageous alternative to prevent and treat PUs, with a recurrence rate of up to 82%. However, the existing clinical guidelines concerning the application of electrical stimulation for PU management and treatment in SCI patients remain limited (Larson et al., 2012; Liu et al., 2014). Thus, an analysis of predictors for PUs could provide valuable information to prevent PU occurrence or recurrence and relieve their severity (Lee et al., 2012).

The endoplasmic reticulum (ER) is the major site for the synthesis and maturation of secreted and transmembrane proteins, activation of the unfolded protein response (UPR), xenobiotic detoxification, and storage of lipid biosynthesis and Ca²⁺ (Fu et al., 2011). ER stress, also known as the UPR, results from an imbalance between the capacity of the ER folding proteins and the client protein load. It is activated by the loss of ER homeostasis, leading to the aggregation of misfolded proteins (Cnop et al., 2012). Protein disulfide isomerase (PDI) is a prototypical member from an extended family of oxidoreductases (ER-resident enzymes) that can catalyze the formation and rearrangement of the posttranslational disulfide bond and can also act as chaperones in protein folding (Muller et al., 2013). PDI has been found in various subcells outside the ER, and it plays a potent role in biological functions on the cell surfaces of lymphocytes, platelets, hepatocytes, and endothelial cells (Jasuja et al., 2012). Binding immunoglobulin protein (BIP)/glucose-regulated protein 78 (GRP78) belongs to the heat shock protein (Hsp70) family and is also a highly abundant ER-resident chaperone. It binds to hydrophobic amino acid stretches, not only stabilizing the immature protein but also keeping it from aggregating with other misfolded or unfolded proteins (Damasceno et al., 2012). Due to consistent pressure on local muscular tissues, ischemia-hypoxia can cause oxidative stress and calcium overload, further resulting in the occurrence of ER stress, where PDI and BIP are induced to prevent target protein or protein substrate from accumulation (Hoozemans et al., 2006). Here, we hypothesize that ER-resident chaperones play crucial roles in the process of PU formation. In this study, we aim to explore the precise mechanism of association between the ER-resident chaperones (PDI and BIP) and PUs.

Materials and Methods

Ethics statement

This study was approved by the Ethical Committee of the Linyi Tumor Hospital. Written informed consent was obtained from all subjects included in this study. If the subject was unable to provide written consent owing to his/her medical condition, proxy consent was instead obtained from the subject's authorized representative and/or his/her legal guardians, and the subject provided written consent for his/her continued research participation as soon as it was possible. This study complied with the guidelines and principles of the Declaration of Helsinki (M, 2014).

Subjects

A group of 67 SCI paraplegia patients with PUs, admitted to the Linyi Tumor Hospital between January 2011 and December 2014, were selected as the PU group. This group included 51 males and 16 females. The patients in the PU group were between 28 and 66 years of age, with a mean age of 50.60 ± 9.15 years, and they were classified as stage I (n = 18), stage II (n = 27), stage III (n = 10), or stage IV (n = 12) according to the National Pressure Ulcer Advisory Panel's updated PU staging system (Black et al., 2007). Additionally, 61 SCI paraplegia patients without PUs were randomly selected as the non-PU group in the Linyi Tumor Hospital during the same period. The non-PU group included 38 males and 23 females ranging in age from 31 to 64 years (mean age: 48.30 ± 9.75 years). The inclusion criteria were as follows: (1) the subject was diagnosed as having a complete SCI (rank A) or an incomplete SCI (rank B) according to the American Spinal Injury Association (ASIA) classification system (Kirshblum et al., 2011), (2) the subject had normal comprehension, with no history of mental illness or cognitive disorders, and subject or one's family members were in the active treatment conditions, and (3) the subject had complete medical data available. Subjects were excluded if he/she (1) presented with several clinical stages of PUs, (2) had potential deep tissue damage or nonstaged PUs, (3) had experienced major stress events, such as losing a spouse or child or suffering from serious illness or surgery, (4) was in critical condition, (5) presented with a combination of diabetes mellitus, cardiovascular disease, and other respiratory diseases, or (6) took anti-inflammatory medicines recently.

Determination of PDI and BIP

Peripheral blood (5 mL) was collected twice (in the PU group, it was collected before debridement and 28 days after debridement, while in the non-PU group, it was collected before the assay) from each subject in the morning after an overnight fast. After being placed at room temperature for 2 h or at 4°C overnight, the blood samples were centrifuged at 1000 g for 20 min, and the supernatant was measured. The enzyme-linked immunosorbent assay (ELISA) kits used for the measurement of the PDI and BIP in both the PU and non-PU groups were obtained from Shanghai Shuangying Biotech Co., Ltd. According to the manufacturer's instructions, the optical density (OD) value was recorded under the single wavelength (450 mm) using an automatic ELISA analyzer. Curve Expert 1.3 software was used to draw a standard curve based on the standard OD values for the measurement of the PDI and BIP.

PU cleaning and debridement

After cleaning the wound with an isotonic saline solution, the aquagel (∼5 mm thick) was placed over the region of necrotic tissue, which then was covered by an isotonic saline solution-containing gauze, which was affixed with a wide hypoallergenic tape. This dressing allows wound exudate to be quickly removed from the wound site, preventing external liquids or bacteria to contaminate the wound site. After being dissolved in water, the necrotic tissues were removed using sterile scissors and forceps. After removal of the necrotic tissues, aquagel was applied, and the tissue was covered by isotonic saline solution-containing gauze. The dressing was changed as described above. When the necrotic tissues were completely removed and the granulation tissue had emerged, the debridement was complete. In addition, the time (day) required for the debridement was observed and recorded.

PU scale for healing score

According to the pressure ulcer scale for healing (PUSH) score (Gardner et al., 2005), the therapist performed dynamic scoring for each PU at 7, 14, 21, and 28 days after debridement. The PUSH score has three parameters, with total score ranging from 0 to 17 points, including the wound area (length × width), exudate volume, and tissue type. When the total score is zero, the wound is healed. The wound area ranged from 0 to 24 cm², with a corresponding score ranging from 1 to 10 points. The exudate volume is classified into four grades, including no volume, a little volume, middle volume, and a great deal of volume, with corresponding scores ranging from 0 to 3 points. The tissue types include intact skin, epithelium, granulation tissue, carious tissue, and necrotic tissue, with a corresponding score ranging from 0 to 4 points.

Data analysis

Data were analyzed using the SPSS 21.0 software package (SPSS, Inc., Chicago, IL). Categorical data are shown as mean ± standard deviation and assessed with the χ² test. For continuous data, t inspection was performed for comparison between two groups, while a one-way analysis of variance was used for comparison between groups, and the least significant difference test was used for pairwise comparisons. Moreover, the Pearson correlation analysis was also used to test the correlation between PDI and BIP in the SCI patients with PUs. The significance was set as a two-tailed p < 0.05.

Results

Baseline characteristics and the levels of PDI and BIP

We hypothesized that the occurrence of PUs is associated with clinical characteristics of SCI patients, which further influenced the expression of PDI and BIP. As shown in Table 1, no significant differences were found in terms of age, gender, smoking history, and education between the PU and non-PU groups (all ps > 0.05). Compared with the non-PU group, the patients in the PU group had a longer period of being bed bound (p < 0.05). The PU group had more patients with rank A and those with a mixture of medullary cone and cauda equina SCI than the non-PU group (both ps < 0.05). The patients in the PU group had higher levels of PDI and BIP than those in the non-PU group (both ps < 0.05). Moreover, the levels of PDI were positively correlated with those of BIP in all SCI patients (r = 0.707, p < 0.0001). These results indicated that the period of being bed bound, severity of SCI, and damage section were associated with the occurrence of PUs after paraplegia, and the PUs upregulated the levels of PDI and BIP.

Table 1.

Baseline Characteristics and the Levels of Protein Disulfide Isomerase and Binding Immunoglobulin Protein of Spinal Cord Injury Paraplegia Patients in the Pressure Ulcer and Non-Pressure Ulcer Groups

Parameters	PU group (n = 67)	Non-PU group (n = 61)	t/χ²	p
Age	50.60 ± 9.15	48.30 ± 9.75	1.377	0.1711
Sex (m/f)	51/16	38/23	1.697	0.0894
Smoking history (yes/no)	39/28	25/36	1.947	0.0516
Bed time	2.44 ± 1.07	1.83 ± 0.79	3.063	0.0027
Education			5.437	0.1424
Primary school	24	19
Junior high school	28	21
High school	15	17
College and above	0	4
ASIA grading			10.650	0.0011
A	39	18
B	28	43
Spinal cord segments			9.936	0.0070
Cone	11	21
Cauda equine	8	13
Mixed cone and cauda equina	48	27
Levels of PDI (pg/mL)	0.086 ± 0.048	0.050 ± 0.005	5.83	<0.0001
Levels of BIP (pg/mL)	2.280 ± 0.664	1.373 ± 0.253	10.02	<0.0001

ASIA, American Spinal Injury Association; BIP, binding immunoglobulin protein; PDI, protein disulfide isomerase; PU, pressure ulcer.

Levels of PDI and BIP and PU staging

We hypothesized that the levels of PDI and BIP were associated with the PU staging. As seen in Table 2, there was no significant difference in the levels of the PDI protein between stages IV and I (p > 0.05). However, stage IV had lower levels of the PDI protein than stages II and III (both ps < 0.01). Additionally, the levels of BIP were lower in stage IV than in stage III PUs (p < 0.01), but there was no significant difference in the levels of BIP between stages IV and II PUs (p > 0.05). In addition, the levels of the BIP were higher in stage IV than in stage I PUs (p < 0.01). The above results indicate that the levels of PDI and BIP first increased and then decreased, depending on the different stages, peaking at stage III. PDI and BIP expression may participate in the regulatory mechanism of ER stress, exhibiting synergistic action.

Table 2.

Levels of Protein Disulfide Isomerase and Binding Immunoglobulin Protein in A Different Pressure Ulcer Staging (x ± SD, pg/mL)

PU staging	PDI	BIP
I (n = 18)	0.050 ± 0.016	1.515 ± 0.163
II (n = 27)	0.096 ± 0.021^a	2.337 ± 0.483^a
III (n = 10)	0.173 ± 0.044^b,c	3.264 ± 0.348^b,c
IV (n = 12)	0.048 ± 0.006^c,d	2.481 ± 0.256^a,c,d

p < 0.01, ^bp < 0.0001 compared with stage I.

p < 0.01 compared with stage II.

p < 0.01 compared with stage III.

Relationship between PUSH scores and PU stages II, III, and IV before and after debridement

We hypothesized that higher PUSH scores were associated with a longer time of debridement, thereby a longer wound surface healing time. As shown in Table 3, the PUSH scores significantly declined at 7 days after debridement for PU stage II (p < 0.01) but exhibited no significant difference between PU stages III and IV at 7 days after debridement (p > 0.05). In addition, the PUSH scores also significantly decreased at 28 days after debridement for PU stages II, III, and IV (p < 0.01). The PU patients in stage III required more time for debridement than those in stage I but required less time than those in stage IV (both ps < 0.01). There was no significant difference in the PUSH scores between stages II and III and between stages III and IV before debridement (both ps > 0.05). The PU patients in stage III had higher PUSH scores than those in stage II but lower PUSH scores than those in stage IV (all ps < 0.01) at 7, 14, 21, and 28 days after debridement. The above results demonstrated that the patients with higher PUSH scores required a longer debridement and a subsequently longer wound surface healing time (p < 0.01).

Table 3.

Pressure Ulcer Scale for Healing Scores on Stages II, III, and IV Before and After Debridement

Staging	II (n = 27)	III (n = 10)	IV (n = 12)	F/t	p
Wound number	39	18	20
Debridement (days)	8.76 ± 4.23	19.58 ± 10.04^a	31.76 ± 12.18^a,b	34.5	<0.0001
PUSH score
Before debridement	7.68 ± 1.92	8.37 ± 2.16	11.89 ± 3.15^a	21.93	<0.0001
7 days after debridement	4.33 ± 1.80^c	7.05 ± 2.31^a	10.72 ± 2.81^a,b	55.32	<0.0001
14 days after debridement	1.16 ± 0.49^c	5.69 ± 2.09^a,c	10.14 ± 2.54^a,b	197	<0.0001
21 days after debridement	0.42 ± 0.12^c	4.42 ± 1.88^a,d	9.52 ± 3.07^a,b	170.8	<0.0001
28 days after debridement	0^c	2.68 ± 1.48^a,d	8.60 ± 2.60^a-c	8.49	<0.0001
F	239.60	22.05	3.80
P	<0.0001	<0.0001	<0.01

PU scale for healing score at 7, 14, 21, and 28 days after debridement compared with PU scale for healing score before debridement: ^cp < 0.01; ^dp < 0.0001.

Stages III and IV compared with stage II: ^ap < 0.01.

Stage IV compared with stage III: ^bp < 0.01.

PUSH, PU scale for healing.

Levels of PDI and BIP in stages II, III, and IV before and after debridement

We hypothesized that the levels of PDI and BIP notably changed in the PU patients after debridement. In Figure 1, it can be seen that the levels of PDI and BIP notably dropped in stages II and III after debridement compared with before debridement (all ps < 0.01). The level of PDI in stage IV increased after debridement but was still lower than that in stage III before debridement. Furthermore, the level of BIP in stage IV decreased after debridement and was lower than that in stage III before debridement (both ps < 0.01). The above results implied that proteins PDI and BIP are involved in the wound healing process in the PU patients.

FIG. 1.

Levels of protein disulfide isomerase (PDI) and binding immunoglobulin protein (BIP) in pressure ulcer stages II, III, and IV before and after debridement. (A-C) Levels of PDI in stages II, III, and IV, respectively; (D-F) levels of BIP in stages II, III, and IV, respectively.

Discussion

Our study evaluated the association between the levels of PDI and BIP and the development and progression of PUs in SCI paraplegia patients. By measuring the PDI and BIP levels in patients with and without PUs and in those before and after debridement, the study demonstrated that the levels of PDI and BIP were associated with the development and progression of PUs. Furthermore, our results suggest that decreased PDI and BIP levels play a potent role in the process of PU healing. One of the findings of our study was that the length of time a patient was bedridden, severity of the SCI, and SCI site influenced the development of PUs. In our study, the patients with PUs were bedridden for a longer period than those without PUs, and the patients with more severe SCI had higher risk of PU formation. Additionally, compared with the 44% of patients without PUs, ∼72% of patients with PUs in our study had a mixture of medullary cone and cauda equina SCI. Consistent with this finding, Stotts and Wu (2007) reported that recovery in patients with PUs is delayed due to an increased length of hospitalization as well as increased healthcare costs. In addition, Wilczweski et al. (2012) demonstrated that critically ill SCI patients had an increased risk of PU formation.

Another finding of this study was that the levels of PDI and BIP were associated with the development and progression of PUs. PU formation is a complex process, commonly resulting from exposure to pressure or shear forces, combined with ischemia, direct cell deformation, and ischemic reperfusion injury in combination with local injury to the skin (Demarre et al., 2013). Interestingly, ER stress has been closely associated with the pathophysiology of organ ischemia as well as reperfusion injury (Liu et al., 2012). The activation of a mechanism, known as the UPR, has been implicated in the reduction of the amount of unfolded or misfolded proteins by increasing the production of the ER chaperones (PDI and BIP/GRP78), optimizing the level of protein folding (Ayala et al., 2012). In our study, the SCI paraplegia patients in the PU group had higher levels of the PDI and BIP than those without PUs. Consistent with the results of our study, Cui found that ER stress-related proteins, including PDI and BIP and their apoptotic pathway, may be implicated in the formation of deep tissue injury of PUs in rats (Cui et al., 2013).

Our results also demonstrated that the levels of PDI and BIP were associated with PU staging. To manage ER stress, cells have developed the UPR to reestablish homeostasis and alleviate ER stress by increasing protein-folding chaperones, such as PDI and BIP. Cells also downregulate the ER protein client load through the inhibition of general protein translation and degradation of misfolded proteins. If the ER stress is prolonged or severe, the UPR promotes programmed cell death (Ozcan and Tabas, 2012). In our study, the levels of PDI and BIP first increased and then decreased, reaching a peak at stage III of PUs. Early in the PU process, due to the consistent pressure on local muscular tissue, ischemia-hypoxia results in oxidative stress and calcium overload, leading to intensive ER stress conditions in which PDI and BIP are induced to prevent target protein or protein substrates from polymerization and eventually protect the cell. Subsequently, with the synergistic reaction between prolonged pressure and reperfusion injury, ischemia-hypoxia worsens, and the number of oxygen free radicals exceeds the capacity of organ handling. Substantial proteins are oxidize modified, and unfolded proteins accumulate, which leads to the consistency or overreaction of ER stress and further decreases the levels of BIP and PDI (Hoozemans et al., 2006). Thus, the levels of PDI and BIP were found to be associated with PU staging.

Interestingly, by calculating PUSH scores for PU stages II, III, and IV before and after debridement, we provide compelling evidence that higher PUSH scores indicated a longer time of debridement and thus a longer wound surface healing time. Hon et al. (2010) found that the PUSH tool is a valid, effective, and evaluative tool for monitoring and documenting the wound progress of PU. In our study, the PUSH scores of patients in stage II significantly declined from 7 to 14 days after debridement, possibly as a result of damage to the dermis, which could be easily debrided through autopepsia (Gunes, 2009). Additionally, in our study, the PUSH scores also remarkably decreased 28 days after debridement for PU stages III and IV. It is therefore entirely possible that stages III and IV refer to deep PUs, largely presenting with full skin loss, even when exposed to bone and muscles (Braga et al., 2013).

Moreover, our study also found that the levels of PDI and BIP largely decreased after debridement. Debridement consists of the removal of the wound's necrotic, dysvascular, and nonviable tissue, leaving behind a red and granular bed. This promotes healing by providing a more responsive acute healing environment (Gordon et al., 2012). After debridement, the PUs gradually heal, and the ER stress tends to be relieved, thus increasing the levels of PDI and BIP and reducing protein folding (Panuncialman and Falanga, 2009).

Taken together, our study provided compelling evidence that ER stress proteins (PDI and BIP) may be involved in the process of PU formation and healing, and the levels of PDI and BIP were associated with the severity of PUs. Our study also provided evidence that PUSH score can be used as a reference to evaluate PU severity and healing. Additional studies with greater sample sizes are needed to determine whether PDI and BIP play a synergistic role in the process of PU healing.

Footnotes

Acknowledgments

We would like to acknowledge the helpful comments from our reviewers.

Author Disclosure Statement

No competing financial interests exist.

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