Decreased expression of Heat Shock proteins may lead to compromised
wound healing in type 2 diabetes mellitus patients
Kanhaiya Singh, Neeraj K. Agrawal, Sanjeev K. Gupta, Gyanendra Mohan, Sunanda Chaturvedi, Kiran Singh
PII:
DOI:
Reference:

S1056-8727(15)00008-2
doi: 10.1016/j.jdiacomp.2015.01.007
JDC 6389

To appear in:

Journal of Diabetes and Its Complications

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Revised date:
Accepted date:

1 December 2014
9 January 2015
11 January 2015

Please cite this article as: Singh, K., Agrawal, N.K., Gupta, S.K., Mohan, G., Chaturvedi,
S. & Singh, K., Decreased expression of Heat Shock proteins may lead to compromised
wound healing in type 2 diabetes mellitus patients, Journal of Diabetes and Its Complications (2015), doi: 10.1016/j.jdiacomp.2015.01.007

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Kanhaiya et al. : Heat shock proteins and impairment of wound healing in T2DM cases

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Decreased expression of Heat Shock proteins may lead to compromised wound healing in
type 2 diabetes mellitus patients

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Kanhaiya Singh1, Neeraj K Agrawal2, Sanjeev K Gupta3, Gyanendra Mohan4, Sunanda

Chaturvedi4 and Kiran Singh1*
Department of Molecular & Human Genetics, Banaras Hindu University,

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Varanasi-221005, India

Department of Endocrinology and Metabolism, Institute of Medical Sciences,

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Banaras Hindu University, Varanasi-221005, India

Department of Surgery, Institute of Medical Sciences, Banaras Hindu University,
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Varanasi-221005, India
Indian Railway Cancer Hospital and Research Centre, N.E.R.,

1* Correspondence

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Kiran Singh

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Varanasi- 221002, India

Department of Molecular & Human Genetics

Banaras Hindu University
Varanasi-221005, India
e-mail: singhk4@rediffmail.com, skiran@bhu.ac.in
Telefax: +91-542-670-2499
Telephone: +91-9454210058

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Abstract
Background: Heat shock proteins (HSPs) are inducible stress proteins expressed in cells

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exposed to stress. HSPs promote wound healing by recruitment of dermal fibroblasts to the site
of injury and bring about protein homeostasis. Diabetic Wounds are hard to heal and inadequate

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HSPs may be important contributors in the etiology of diabetic foot ulcers (DFU).

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Objective: To analyze the differential expression of HSPs and their downstream molecules in
human diabetic wounds compared to control wounds.

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Methods: Expressional levels of HSP27, HSP47 and HSP70 and their downstream molecules
like TLR4, p38-MAPK were seen in biopsies from 101 human diabetic wounds compared to 8

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control subjects without diabetes using RT- PCR, western blot and immunohistochemistry.

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Results: Our study suggested a significant down regulation of HSP70, HSP47 and HSP27 (p

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value = <0.001 for HSP70; p value = 0.007 for HSP47; p value = 0.007 for HSP27) in DFU
along with their downstream molecules TLR4 and p38-MAPK (p value = 0.006 for p38-MAPK;

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p value = 0.02 for TLR4). HSP70 levels were significantly lower in male subjects and their
levels increased significantly with the grades of wound on Wagners scale. Infection status of the
wounds was found to be significantly associated with the increased levels of HSP70 and HSP27
in infected diabetic wounds.
Conclusions: Our study demonstrates that the down regulation of HSPs in diabetic wounds is
associated with wound healing impairment in T2DM subjects.
KEY WORDS: Heat shock proteins; Wound healing impairment; type 2 diabetes mellitus
(T2DM); TLR4; MAPK; Diabetic foot ulcer

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Introduction
Heat shock proteins (HSPs) are heterogeneous gene products of a highly conserved family of

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stress proteins [1]. Their molecular weights vary roughly from 16 KDa to 110 KDa, and they are
rapidly expressed in cells exposed to a variety of stress [2]. All organisms ranging from

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archeabacteria to eubacteria, or from plants to animals, respond to endogenous or exogenous

stress by inducing HSPs [1]. The synthesis and expression of these stress proteins is regulated

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mainly by a transcription factor known as heat shock factor-1 (HSF-1) which bind to the heat

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shock elements (HSE) present in the promoter region of specific genes. In addition to this
emergency response, they also serve as molecular chaperones in various physiological and

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pathological conditions by bringing about the folding of nascent polypeptides and targeting
improperly folded proteins for degradation [3]. These proteins maintain a state of homeostasis

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during normal cell growth as well as in pathological condition by maintaining the cellular

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integrity [4]. Recent reports have suggested that HSPs after their release in the blood, also
participate in signal transduction [5]. These proteins have anti-inflammatory properties as they

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inhibit nitric oxide synthase activity along with NF-B dependent gene expression [6]. Emerging
evidences also suggest certain members of HSPs participate in both innate and adaptive
immunity [7]. HSPs can modulate cellular adaptive response by modulating CD8+ cytotoxic cell
receptor on one hand and can directly stimulate innate immune response by toll like receptor
(TLR) mediated signaling on the other [5, 8].
Wound healing is a dynamic process which includes 4 main overlapping stages ranging from (i)
acute inflammation through (ii) fibrin rich exudates organization, (iii) re-epithelialization to (iv)
granulation tissue formation [9]. The healing wound bed contains different inducible HSPs like
HSP90, HSP70, HSP47 and HSP27 which all together bring about protein homeostasis and cell
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proliferation during wound healing [10]. HSP90 brings about wound healing by supporting the
differentiation of keratinocytes [11]. HSP70 lowers oxidative damage of fibroblasts and supports

cell proliferation in the wound area by inhibiting stress induced apoptosis and TLR activation

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[12]. HSP70 is also a ligand for TLR4, which is shown to be an important contributor to wound
closure [13]. The functioning of HSP70 is aided by expression of HSP47, which is

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predominantly involved in pro-collagen synthesis and binding with collagen type II and III [14].

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Small HSPs like HSP27 also supports wound healing by stabilizing actin microfilaments,
supporting endothelial cell migration in the wound bed, protecting sensory neuron degeneration

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and inhibiting stress induced apoptosis [15]. This function of HSP27 is governed by its MAPK

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mediated phosphorylation [16].

Type 2 diabetes mellitus (T2DM) is characterized by chronic hyperglycemia and is a proximal

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determinant of secondary complications like neuropathy, tissue ischemia and infection [17]. The

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wounds in T2DM patients often turn into non healing micro-environment leading to chronic
ulcers due to less oxygen supply, death and deformity in migrating fibroblasts, abnormal matrix

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degradation and oxidative stress [18, 19]. T2DM also regulates the HSP synthesis by modulation
of transcriptional or translational processes [20]. Deficient insulin signaling in diabetic
individuals lead to glycogen synthase kinase 3- (GSK-3) mediated inhibitory phosphorylation
and deactivation of HSF1.This result in the low levels of intracellular HSPs (iHSPs) especially in
insulin sensitive tissues like muscles and liver which further interfere with insulin signaling via
activation of inflammatory cytokines, c-Jun N-terminal kinase (JNK) and IkappaB kinase (pIKK) in a cyclic manner [4]. This decreased level of iHSPs create an impaired stress response in
diabetic individuals manifested by glycation, oxidation and aggregation of cellular proteins
which in majority of cases lead to the disruption of homeostatic processes[4].
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literature supports the deficiency of HSPs especially HSP70 or their transcriptional activators in
diabetic wounds in various model systems [21] but little is known about the role of HSPs in

human diabetic wounds. In the present work we have tried to see the expression levels of HSP27,

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HSP47 and HSP70 and their downstream molecules like TLR4, p38 mitogen-activated protein
kinase (p38-MAPK) in biopsies from human diabetic wounds in comparison to non diabetic

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wounds.

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Materials and methods:

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Subjects:

This case control study comprised of 109 subjects in which 101 were Diabetic foot ulcer (DFU)

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cases and 8 were controls without having T2DM. All DFU patients included were Diabetics who

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had non-healing wounds of > 4 weeks duration, thus qualifying as Diabetic wounds. Majority of
the patients had lower extremity wounds 90% of which were located on the foot alone and in the

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remaining 10% foot + lower leg were involved. Both the plantar and dorsal aspects of the foot

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were involved in the majority of the cases. The samples were collected at the time of their (the
patients) first visit to the Diabetic foot clinic. Samples were taken from the wound margins
during the debridement process and the histological analysis was performed to determine the cell
types (Sup. Figure1). Classification of wounds was made on the basis of the Wagners Grading
System [22]. The presence and absence of infection in the wounds were also recorded. Samples
were collected from the OPD clinics and operation theatres of Department of Endocrinology and
Metabolism and Department of Surgery, Institute of Medical Sciences, Banaras Hindu
University, Varanasi, India during the period of July 2010 to December 2013. Tissue samples
were collected in RNAlater solution (P/N AM7020, Ambion, Inc., Austin, TX, USA) and

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phosphate buffer saline (PBS) for RNA and Protein isolation respectively and kept frozen at 80C until use. For immunohistochemical staining, samples were collected in Boiun's fixative

solution and kept at room temperature. Patients underwent a standardized clinical and laboratory

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evaluation. The T2DM patients having neuropathic, vascular or traumatic ulcers were included in
this study. Screening for neuropathy was done by taking a history of sensory loss and other

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symptoms such as a burning sensation or paresthesias. Clinical neurological examination

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included the assessment of the vibratory threshold perception using a 128 Hz tuning fork and
assessment of pain and fine touch with a pin and 10g monofilament respectively. The tendon

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reflexes and muscle power were measured in patients with sensory neuropathy. Screening for
vascular involvement included a detailed history of vascular insufficiency, clinical examination

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for signs of chronic ischemia and assessment of all lower limb pulses. A bed side hand held

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Doppler study was carried out in all clinically suspicious cases and ABPI (ankle brachial
pressure index) of < 0.9 was considered indicative of peripheral vascular disease. Age and sex

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matched control tissues were obtained by full thickness wound biopsies of post cellulitic chronic

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ulcers of the foot and the distal leg. These were non-healing ulcers present for 4 weeks or more
following cellulitis of the lower limb. Each patients family history, habits (smoking, alcoholism
etc.), and disease were recorded through a questionnaire (Table 1). The exclusion criteria of the
study included presence of co morbid disorders such as thyroid dysfunction and patients not
belonging to north India. The study was approved by the Institutional Human Ethics Committee
of Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. Informed written
consent was obtained from every participant of each group.

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Semi-quantitative RT-PCR
Total RNA was isolated from wounds samples using TRIzol reagent followed by DNase

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treatment. cDNA was synthesized and semi-quantitative RT-PCR analysis of HSP70, HSP47
HSP27, p38-MAPK and TLR4 was done in 101 DFU cases and 8 controls. Sequences of the

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implicated primers in the study are provided in Table 2. The PCR conditions were initial
denaturation step of 94C for 5 min followed by 30 cycles of 30 sec at 94C, 40 sec at 58C, 40

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sec at 72C and then a final extension step of 10 min at 72C. Glyceraldehyde 3-phosphate

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dehydrogenase (GAPDH) expression level was checked as an internal control to ascertain the
quality of cDNA. Expression of gene transcripts were quantied after normalizing samples using

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Quantitative Real-time PCR

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GAPDH gene.

A quantitative RT-PCR (RT-qPCR) experiment was also performed to validate the results

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obtained by semi-quantitative RT-PCR in 88 DFU samples of different grades on Wagner Scale

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and 8 controls. RT-qPCR experiment was performed according to the manufacturers protocol
(Applied Biosystem) using primers for HSP27, HSP47, HSP70 and GAPDH. Briey, 20 l total
reaction volume containing 10 l SYBR Green, 0.1 l each forward and reverse primer (10pm/
l) and 2 l cDNA was used in PCR using ABI 7500 instrument. PCR was performed with an
initial incubation at 50C for 2 min, then followed by 10 min denaturation at 95C and 40
cycles at 95C for 15 s, 60C for 1 min and 72C for 15 s. Gene expression profiles were
normalized to the mRNA levels of housekeeping gene GAPDH. CT and the relative fold
change of HSPs in DFU cases were calculated according to our previous report [23].

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Immunohistochemical staining
Wound tissue samples obtained during the debridement process were fixed in Bouins solution,

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embedded in paraffin, and sectioned into 3 m thick sections. 5 DFU wounds and 5 control
wounds were randomly selected for the study. Anti HSP27 antibody (Catalog No. ab5579,

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Abcam Inc., Cambridge, MA., 1:75 in PBS), Anti HSP47 antibody (Catalog No. ab88115,
Abcam Inc., Cambridge, MA., 1:75 in PBS) and Anti HSP70 antibody (Catalog No. ab47455,

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clone C92F3A-5, Abcam Inc., Cambridge, MA., 1:75 in PBS) were applied separately to the

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deparaffinized sections and incubated in a wet chamber at 4C for 12 hours. Vectastain Elite
ABC Kit (Vector Laboratories, Burlingame, CA) was used for immunohistochemical staining.

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Slides were counterstained with Hematoxylin (Himedia, India). Cells having brown-stained
cytoplasm were regarded as positive. Similar staining time and procedure was adopted for all

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tissue samples. Expression patterns of HSP27, HSP47 and HSP70 in control and diabetic wounds

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were done under the microscope (Nikon) using different magnifications (4, 10, 20 and 40).
Documentation of acquired images was done using a calibrated digital camera system (Nikon

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eclipse 80i) together with the software evaluation package (NIS Elements software). The
expression density HSPs in wound biopsies were computed according to a previous study by
Souil et al. [24].
Western blot
Western blot analysis was performed for HSP27, HSP47 and HSP70 on whole-tissue extracts of
wound biopsies to verify the results of IHC. About 50 g of protein was loaded on 12 % SDSPAGE gel, which was transferred to nitrocellulose membrane and then blocked with 5% of skim
milk in TBS. For HSP27, rabbit polyclonal anti-HSP27 antibody (Catalog No. ab5579, Abcam

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Inc., Cambridge, MA., 1:1000 dilution), for HSP47, mouse polyclonal anti-HSP47 antibody
(Catalog No. ab88115, Abcam Inc., Cambridge, MA., 1:1000 dilution) and for HSP70, mouse

monoclonal anti-HSP70 antibody (Catalog No. ab47455, clone C92F3A-5, Abcam Inc.,

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Cambridge, MA., 1:1000 dilution) were used and then incubated with the secondary antibody
linked to horseradish peroxidase. The immunoreactive bands were visualized by the Enhanced

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Chemiluminescence System (Amersham Biosciences). Blots were stripped off and reprobed with

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anti-GAPDH antibody.

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Statistical analysis

The data were expressed as mean considering standard error of mean as error bars. Statistical

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signicance (P < 0.05) was determined by Students t test (two-tailed) and nonparametric

Results

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Statistics 20.0 software.

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ANOVA. Statistical analysis of data was performed using Graph Pad Prism 5.01 and IBM SPSS

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Semi-quantitative RT-PCR analysis showed that there was significant down regulation of
HSP70, HSP47 and HSP27 in wounds of DFU patients compared to control cases (p value =
<0.001, t = 5.59, R squared = 0.22 for HSP70; p value = 0.007, t = 2.77, R squared = 0.06 for
HSP47; p value = 0.007, t = 2.72, R squared = 0.06 for HSP27) (Figure 1, Sup. Figure 2). This
down-regulation of analyzed HSPs message was again confirmed by RT-qPCR analysis (p-value
< 0.001, mean fold change = 2.47 0.15, t = 4.73 for HSP70; p-value < 0.001, mean fold change
= 2.04 0.14, t = 4.17 for HSP47; p-value = 0.03, mean fold change = 2.79 0.15, t = 2.10 for
HSP27) (Figure 1.b). The mRNA level of HSP70 was higher in females as compared to males
(p-value = 0.017, t = 2.42, R squared = 0.06) while the expressions of HSP47 and HSP27
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message were similar in both genders (p-value = 0.63, t = 0.47, R squared = 0.002 for HSP47
and p-value = 0.81, t = 0.23, R squared = 0.001 for HSP27) (Figure 2). HSP70 and HSP27 were

found to be significantly higher in the infected diabetic wounds compared to controls (p-value =

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0.012, t = 0.2.55, R squared = 0.06 for HSP70 and p-value = 0.03, t = 2.17, R squared = 0.045
for HSP27) while HSP47 levels remained unaltered even in the presence of infection (p-value =

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0.70, t = 0.38, R squared = 0.001 for HSP47) (Figure 3). HSP70 level was found to increased

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significantly with the severity of diabetic wounds on Wagners scale (p-value = 0.01, t = 3.79, R
squared = 0.105) while other two HSPs levels remained unaltered with the severity of diabetic

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wounds (p-value = 0.26, t = 1.35, R squared = 0.041 for HSP47 and p-value = 0.24, t = 1.42, R
squared = 0.04 for HSP27) (Figure 4, Sup. Figure 3). Analysis of mRNA downstream molecules

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to HSP27 and HSP70 were also done.

Expression of p38-MAPK suggested its similar

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expression patterns as HSP27 (Figure 5 a, 5 b, Sup. Figure 4) and TLR4 expression were similar
to the patterns of HSP70 (p value = 0.02, t = 2.30, R squared = 0.05 for TLR4; p value = <0.001,

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t = 5.59, R squared = 0.22 for HSP70) (Figure 6 a, 6 b, 6 c, Sup. Figure 5). Western blot analysis

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showed that these HSPs were significantly down regulated at translational levels also (p value =
0.001, t = 3.72, R squared = 0.42 for HSP70; p value = <0.0001, t = 5.88, R squared = 0.65 for
HSP47; p value = 0.01, t = 2.77, R squared = 0.31 for HSP27) (Figure 7). Figure 8.A shows the
positive and negative (no primary antibody) staining patterns of different antibodies of HSPs
used in IHC study. Absence of any signal in negative controls suggested that the signal in
positive control was specific signal of primary antibody binding to the antigens.
Immunohistochemical expression analysis between groups also suggested significant down
regulation of HSP70, HSP47 and HSP27 between the wound biopsies of DFU cases and controls

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(Figure 8.B, 8.C, 8.D). The down regulation of TLR4 in DFU cases compared to control wounds
was in a similar pattern to HSP70 (Figure 9, Sup. Figure 6)

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Conclusion

The cells of all organisms generally counteract sub lethal endogenous or exogenous stresses by

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activating transcription of a specific set of genes termed as HSPs. The stresses may be

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physiological (e.g., inflammation, ischemia), pathological (e.g., bacterial or viral infection) or

environmental (e.g. heat shock, oxidative stress or heavy metal poisoning) [25]. Some HSPs are

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normally expressed in cells regulated by hormones like estrogens while others are produced
exclusively during stress periods by certain cells [26]. A cell dependent unique pattern of

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induction and repression of other genes is followed after expression of HSPs. These HSPs are
highly conserved in amino acid sequences and contain a specific DNA motif called heat shock

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element (HSE) [27]. These HSEs when occupied by HSFs bring about the transcription of HSPs.

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HSPs are also shown to be protective against insulin resistance and obesity induced

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inflammation, thereby abrogating T2DM [28]. Some members of HSPs like HSP90 and HSP70
are also shown to promote wound healing by promoting recruitment of dermal fibroblasts and
endothelial cells to the site of injury, thereby, supporting re-epithelialization in model animals
[29]. Wounds of T2DM patients are hard to heal and inadequate amount of inducible HSPs may
be one of the important contributors in the etiology of DFU [30]. The present work was designed
to correlate the expression levels of three HSPs namely HSP70, HSP47 and HSP27 along with
their downstream interacting partners like p38-MAPK and TLR4 with wound healing
impairment in human T2DM subjects. Several contributing factors like gender of the subjects,
infection status of the wounds and severity of wounds on Wagners scale were also taken into
account.
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Immediate release of anti-apoptotic and anti-oxidative HSP70 by keratinocytes prevailing in the
epidermis is a prerequisite of an acute wound [31]. This HSP70 after secretion in the wound

microenvironment bring about the regulation of the inflammatory phase by coordinating pro and

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anti-inflammatory responses [32]. Appropriate levels of HSP70 in wounds often lead to abundant
granulation tissue synthesis and proper healing. The analysis of our data indicated that there was

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significant down regulation of HSP70 in human diabetic wounds compared to non diabetic

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wounds. Our result is supported by the findings of Oberringer et al. (1995), according to which
non-diabetic wounds lacking adequate HSP70 proteins may often develop into chronic decubitus

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ulcers [33]. Another report by Vgh et al. (1997) suggested that the application of Bimoclomol, a
strong inducer of HSP70, significantly enhance wound closure in thermally wounded STZ

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diabetic rats compared to controls [34]. Another finding that external HSP70 inductions in

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chronic wounds of streptozotocin-induced diabetic mice bring about the rapid closure of wounds
also supported our hypothesis that decreased levels of HSP70 is one of the causes of DFU in

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humans [35]. Lack of proper activation of innate immunity by TLR4 mediated signaling is also

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one of the causes of developing DFU [23, 36, 37]. HSP70 being a ligand for TLR4 is also
required in proper amount in healing wounds [13]. Hence we simultaneously analyzed the
expression levels of HSP70 and TLR4 in DFU subjects and the results obtained indicated that
less expression of HSP70 and TLR4 combined together bring about compromised wound healing
in them. Levels of HSP70 and TLR4 both showed a gender dependency as female subjects had
comparatively higher amount of HSP70 and TLR4 compared to their male partners. Regulation
of HSP70 and TLR4 via estrogen is the reason behind this finding and it supports the higher
incidence of DFU in males compared to females [38, 39]. The levels of HSP70 along with TLR4
were found to be dependent on the grades of wounds and severe wounds were associated with

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higher levels of HSP70 compared to normal wounds. The levels of HSP70 were also found to be
significantly associated with the infection status of the diabetic wounds with higher levels of

HSP70 transcripts in infected diabetic wound compared to non infected wounds. The reason for

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this observation may be the finding that microbial infections bring about an increase in

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expression of HSP70 in infected cells [40].

HSP27 plays an important role in modulating actin dynamics in response to various stimuli.

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HSP27 participates in the wound healing process by regulating fibroblasts and endothelial cell

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migration, adhesion and invasion in the wound microenvironment. The expression and spatial
distribution of HSP27 is shown to regulate normal wound healing [41]. This effect of HSP27 is

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mainly orchestrated by p38MAPK mediated phosphorylation. HSP27 is only known substrate of

certain mitogen activated protein kinase-activated protein kinase (MAPKAPK) 2/3 [42, 43].

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HSP27 is also a protective agent against diabetic neuropathy and hence diminished expression of

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HSP27 may also compromise healing in diabetic neuropathic ulcers [44]. The findings of present
work suggested a significant down regulation of HSP27 both at the transcriptional and

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translational levels in biopsies of DFU cases compared to controls. The p38-MAPK expression at
transcription levels also followed the similar trend in DFU subjects. This finding of ours was in
coordination with a recent report by Crowe et al. (2013) which suggests that mice deficient in
orthologue of HSP27 display delayed wound healing by lesser collagen deposition and prolonged
inflammatory stage [45]. HSP27 levels like HSP70 was also dependent upon the infection status
of diabetic wounds. This finding is supported by the observation of Wainberg et al. that
cumulative expression of HSP70 and HSP27 by CD4+ lymphocytic cells shoot up following
acute infection of DNA and RNA mediated viruses [46]. The expressional levels of HSP27

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transcripts were found not be associated with grades of wounds and the gender of the DFU
subjects.

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HSP47 is also called collagen binding protein 1 and is a molecular chaperone specific for
procollagen (47). Being localized to endoplasmic reticulum, the function of HSP47 is to bring

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about the maturation of collagen by mediating post-transcriptional modification in procollagen

[48]. Wang et al. (2002) shown that HSP47 is inducible in wounds and is one of the positive

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contributors in healing fetal wounds [49]. Our study showed that HSP47 expression was

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significantly lower in diabetic wounds compared to control wounds. This down-regulation of

HSP47 in diabetic wounds may abrogate wound healing by inappropriate collagen synthesis and

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less differentiation of fibroblasts into myofibroblasts [50]. This finding of ours is supported by
the report of Wang et al. (2009) which supports the wound healing enhancer capability of HSP47

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in alloxan-induced diabetes rats [51]. Unlike HSP70 and HSP27, HSP47 transcripts did not show

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any gender, wound grade or wound infection dependency. One limitation of the present study
was the less number of control samples compared to DFU cases, reason being unwillingness of

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controls to provide tissue biopsy due to pain and fear associated with the process.
In conclusion, our study demonstrates the combined down regulation of HSP70, HSP27 and
HSP47 expression in diabetic wounds creates a sort of hostile microenvironment in the wound
bed and abrogates wound healing in T2DM subjects by less collagen availability, improper
migration of wound healing fibroblasts, sensory neuropathy promotion and persistent
inflammation. Further research is required to see the effect of activators of HSPs on healing
patterns of diabetic wounds in vitro for effective therapeutic intervention in this respect.

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Conflict of interest
The authors declare no conflict of interest.

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Funding

This work was funded by Department of Science and Technology, New Delhi, India (SR/FT/LS-

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101/2010). Financial assistance by Department of Biotechnology, Ministry of Science and

Technology, New Delhi, India in form of Senior Research fellowship to the first author is

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thankfully acknowledged. We thank Nilu Prasad and Shanti Besra, Laboratory Superintendents,

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Indian Railway Cancer Hospital and Research Centre, N.E.R., Varanasi, for their technical
assistance during IHC work.

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Author contribution

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Kanhaiya Singh designed research, performed experiments, collected and analyzed the data,
wrote the paper. Kiran Singh designed research, interpreted data, and wrote the paper. N.K.A.,

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S.K.G., G.M. and S.C. provided samples and did the clinical evaluation of patients. Their critical

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comments helped us in writing and approving the final manuscript. Dr. Kiran Singh is the
guarantor of this work, had full access to all the data, and takes full responsibility for the
integrity of data and the accuracy of data analysis.

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Figure Legends:
Figure.1 RT-PCR analysis indicates the decreased expression of HSP70, HSP47and

HSP27 transcripts in DFU patients. RNA was isolated from wound samples of DFU

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patients and Controls, cDNA was synthesized and RT-PCR for HSP70, HSP47, HSP27
and GAPDH was performed.

(A) Bar Graph represents the percent ratio which was calculated for the expression of

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HSPs and GAPDH in Controls and DFU respectively. Expression of HSP70, HSP47 and
HSP27 transcripts were found to be downregulated in DFU cases as compared to controls

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(p value = <0.0001, t = 5.59, R squared = 0.22 for HSP70; p value = 0.007, t = 2.77, R
squared = 0.07 for HSP47; p value = 0.007, t = 2.72, R squared = 0.06 for HSP27.

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(B) Bar Graph of qPCR analysis showing the lower expression of HSPs mRNA in the
wounds of DFU patients. Analysis was done in 88 DFU cases and 8 controls. Fold
change in the expression of genes was determined using the CT method of relative

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quantification. The graph was plotted using log (Relative average fold change) i.e. (log 2CT

). The graph clearly showed that HSP70, HSP47 and HSP27 were down regulated

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significantly in the wounds of T2DM cases compared to controls (p-value < 0.001, mean
log (fold change) = - 2.47 0.15, t = 4.73 for HSP70; p-value < 0.001, mean (log fold)

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change = - 2.04 0.14, t = 4.17 for HSP47; p-value = 0.03, mean log (fold change) = -

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2.79 0.15, t = 2.10 for HSP27). Log (10) = 1; log (100) = 2.

Figure2. Bar Graph showing the correlation of HSP70, 47 and 27 transcripts with
the Gender of DFU patients. Among the diabetic wounds, males showed relatively
lesser amount of HSP70 mRNA transcripts with respect to females (p-value = 0.017, t =
2.42, R squared = 0.06) while the expressions of HSP47 and HSP27 message were
similar in both genders (p-value = 0.63, t = 0.47, R squared = 0.002 for HSP47 and pvalue = 0.81, t = 0.23, R squared = 0.001 for HSP27).

Figure3. Bar Graph showing the correlation of HSP70, 47 and 27 transcripts with
the infection status of DFU wounds. HSP70 and HSP27 were found to be significantly
associated to the infection status of the diabetic wounds. Infected wounds contained
higher levels of HSP70 and HSP27 compared to sterile wounds (p-value = 0.012, t =
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0.2.55, R squared = 0.06 for HSP70 and p-value = 0.03, t = 2.17, R squared = 0.045 for
HSP27). HSP47 levels remained unaltered even in the presence of infection (p-value =

0.70, t = 0.38, R squared = 0.001 for HSP47).

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Figure4. Bar Graph showing the comparison of HSP70, 47 and 27 transcripts with
the wound grades on Wagners scale. HSP70 message was significantly dependent on

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the grade of wounds on Wagners scale and its level were high in more severe wounds
compared to less severe wounds (p-value = 0.01, t = 3.79, R squared = 0.105). HSP27

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and HSP47 levels remained unaltered with the severity of diabetic wounds (p-value =
0.26, t = 1.35, R squared = 0.041 for HSP47 and p-value = 0.24, t = 1.42, R squared =

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0.04 for HSP27).

Figure5. Bar graph showing p38-MAPK having similar expression patterns as

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HSP27. (A) Expression of p38-MAPK and HSP27 transcripts were found to be down
regulated in DFU cases as compared to controls (p value = 0.006, t = 2.79, R squared =

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0.07 for p38-MAPK; p value = 0.007, t = 2.72, R squared = 0.06 for HSP27). (B) p38MAPK message was significantly dependent on the grade of wounds on Wagners scale

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(p-value = 0.02, t = 3.34, R squared = 0.11) and shared a similar pattern followed by
HSP27 transcripts in DFU wounds.

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Figure6. Bar graph showing TLR4 having similar expression patterns as HSP70. (A)
Expression of TLR4 and HSP70 transcripts were found to be significantly down
regulated in DFU cases as compared to controls (p value = 0.02, t = 2.30, R squared =
0.05 for TLR4; p value = <0.001, t = 5.59, R squared = 0.22 for HSP70). (B) Both
HSP70 and TLR4 message were found to be sharing a similar patterns dependent upon
grade of wounds on Wagners scale. (C) The mRNA levels of both HSP70 and TLR4
were significantly higher in females as compared to males (p-value = 0.017, t = 2.42, R
squared = 0.06 for HSP70; p value = 0.02, t = 2.3, R squared = 0.06).

Figure7. HSPs protein expression was analyzed in 15 DFU patients and 6 controls.
Tissue samples collected from control {C} and DFU patients {P} were homogenized and
Western blot analysis was performed for expression of HSP70, HSP47, HSP27 and
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GAPDH protein. Bar Graph showing down regulation of HSPs in wounds of DFU
patients compared to controls (p value = 0.001, t = 3.72, R squared = 0.42 for HSP70; p
value = <0.0001, t = 5.88, R squared = 0.65 for HSP47; p value = 0.01, t = 2.77, R

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squared = 0.31 for HSP27).

Figure8. Immunohistochemistry of HSP70, 47 and 27 in wound samples with insets

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showing detail of staining (40 X magnifications). Microwave-induced antigen retrieval

used 0.01 M citrate buffer (pH 6). Figure 8.A shows the positive and negative (no

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primary antibody) staining patterns of different antibodies of HSPs used in IHC study.
Absence of any signal in negative controls suggested that the signal in positive control

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was specific signal of primary antibody binding to the antigens. Immunohistochemical

expression analysis among groups also suggested significant down regulation of HSP70,
HSP47 and HSP27 between the wound biopsies of DFU cases and controls (Figure 8.B,

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8.C, 8.D).

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Figure9. Immunohistochemistry of TLR4 in wound samples with insets showing

detail of staining (40 X magnifications) with Mouse monoclonal anti-TLR4 antibody.

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Upper panel shows the negative (no primary) (left) and positive staining (right). Lower
panel shows the Immunohistochemistry for TLR4 in non-diabetic control wound (left)

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and diabetic wound (right). TLR4 was found to be down regulated in DFU cases
compared to control wounds in a similar pattern to HSP70.

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Table 1 Biochemical and Demographic parameters of DFU patients (N = 101) and
controls (N = 8). Data are presented as mean SD or as number (percentage).

Age in years; mean SD

DFU (N = 101)
54.22 8.94 years

BMI in kg/m2; mean SD

21.69 2.36 Kg/m

23.45 1.95

0.15

Duration of T2DM in years; mean SD

10.18 4.46 years

N/A

--

Male

69 (68.32 %)

5 (62.5%)

0.88

Female

32 (31.68 %)

3 (37.5 %)

0.81

10.6 (8.7 to 13) %

N/A

--

13 (12.87 %)

N/A

--

30 (29.70%)

N/A

--

PARAMETERS

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HbA1c levels (%) (Mean, range)

Nephropathy present (Serum creatinine > 1.4 mg/dl); n (%)

Neuropathy present (by monofilament test); n (%)

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Hypertension present (systolic BP > 140 mm of Hg); n (%)

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Family history present; n (%)

p-value
0.61

61(60.39 %)

N/A

35(34.65 %)

N/A

--

11 (10.89%)

N/A

--

Dislipidimea present (Serum cholesterol and Tgy levels > 200

mg/dl); n (%)

15(14.85 %)

N/A

--

Infection present (Wound culture positive for microbes); n (%)

56(55.44 %)

N/A

--

36 (35.64 %)

N/A

--

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Bone involvement (Osteomyelitis); n (%)

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Retinopathy present; n (%)

Control (N = 8)
56.27 3.42

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Table 2: Primers used for RT-PCR analysis of different genes studied and base pair products of

Reverse Primer

Amplicon size
(bp)

Forward Primer

ACCAAGCAGACGCAGATCTTC

CGCCCTCGTACACCTGGAT

HSP27

TCCCTGGATGTCAACCACTTCG

GGGACAGGGAGGAGGAAACTTG

HSP47

CGCCATGTTCTTCAAGCCA

CATGAAGCCACGGTTGTCC

70

ATGCCGAAGATGAACTTTGC

TCTTATCTGAGTCCAATACAAGCATC

94

CAGAGTTTCCTGCAATGGATCA

GCTTATCTGAAGGTGTTGCACAT

85

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TLR4

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p38-MAPK

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HSP70

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the amplicons.

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