lncRNA MALAT1 Accelerates Wound Healing of Diabetic Mice Transfused with Modified Autologous Blood via the HIF-1α Signaling Pathway.

Impaired wound healing is a debilitating complication of diabetes. The long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been recognized to be differentially expressed in various diseases. However, its underlying mechanism in diabetes has not been fully understood. Notably, we aim to examine the expression of MALAT1 in diabetic mice and its role in wound healing involving the hypoxia-inducible factor-1α (HIF-1α) signaling pathway with a modified autologous blood preservative solution reported. A mouse model of diabetes was established. MALAT1 was identified to promote the activation of the HIF-1α signaling pathway and to be enriched in autologous blood through modified preservation, which might facilitate the improvement of physiological function of blood cells. Through gain- or loss-of-function approaches, viability of fibroblasts cultured in high glucose, wound healing of mice, and collagen expression in wound areas were enhanced by MALAT1 and HIF-1α. Taken together, the present study demonstrated that the physiological status of mouse blood was effectively improved by modified autologous blood preservation, which exhibited upregulated MALAT1, thereby accelerating the fibroblast activation and wound healing in diabetic mice via the activation of the HIF-1α signaling pathway. The upregulation of MALAT1 activating the HIF-1α signaling pathway provides a novel insight into drug targets against diabetes.

Introduction

Diabetes remains one of the major threats to human health and affects millions of people across the world. Characterized by autoimmune destruction of insulin-producing cells, type 1 diabetes causes hyperglycemia and abnormal glucose metabolism, whereas type 2 diabetes is manifested with insulin resistance and progressive β cell dysfunctions. Hyperglycemia is considered to be a primary manifestation of diabetes and is caused by defects in insulin secretion and/or insulin action. Hyperglycemia can damage various organs, including the eyes, kidneys, nerves, heart, and blood vessels. Previous research found that diabetes is responsible for delayed wound healing and the formation of minor skin wounds, non-healing ulcers, infection, gangrene, or amputation. However, the prognosis for chronic wounds remains poor even with the advent of various treatments, such as tight glucose control and meticulous wound care, thus leading to an urgent need for more effective interventions of chronic wound healing.

Previous investigations have shown that long non-coding RNAs (lncRNAs) are involved in diverse cellular processes, including immune responses chromosome dynamics, imprinting control, and circuitry control of human pluripotency. In addition, a wide array of long non-coding RNAs (lncRNAs) are involved in disease etiology.7, 8, 9 lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in endothelial cell function and vessel growth, whereas MALAT1 silencing induces a promigratory response and enhances basal sprouting and migration. In addition, a previous study suggested that MALAT1 could serve as a new biomarker for the prediction of gestational diabetes. The above information and findings led to a hypothesis that MALAT1 could manifest itself as a novel biomarker in diabetes. However, the detailed molecular mechanisms underlying diabetes remain to be elucidated. As a heterodimeric transcription factor, hypoxia-inducible factor-1α (HIF-1α) plays a protective role in β cells after islet transplantation of human and mouse islets.12, 13 Apart from these findings, the function and differentiation of myeloid-derived suppressor cells were altered by HIF-1α in the tumor microenvironment. In this study, we aim to explore the function of MALAT1 in autologous blood samples treated by an ordinary (termed as ordinary blood) or a modified (termed as modified blood) preservative, respectively. Due to the presence of a certain amount of mannitol, the solution of modified preservative was shown to improve the physiological status of blood by stabilizing the permeation of red blood cells (RBCs). It has also been shown that a reduced sugar level can prevent the accumulation of advanced glycation end products on the erythrocyte membrane so as to maintain the integrity of the erythrocyte membrane and to improve its oxygen-carrying capability in diabetic mice. This study further demonstrated the specific role of MALAT1 in inducing fibroblast activation and wound healing in diabetic mice. In addition, the underlying mechanism of MALAT1 involves the HIF-1α signaling pathway.

Results

Modified Autologous Blood Preservatives Elevated the Expression of MALAT1 in Fibroblasts

Flow cytometry was employed in order to determine platelet aggregation after the whole blood samples of mice were preserved in ordinary and modified preservatives. The results indicated that the blood platelet aggregation rate was significantly higher in the improved group (mice treated with the modified preservative solution) than that in the ordinary group (Figure 1A, p < 0.05), indicating that platelet aggregation of blood was lower when blood samples were preserved using the modified preservative solution. In addition, blood physiological indicators were detected using an automated hematology analyzer. The results showed that the levels of blood glucose (BG), glycosylated hemoglobin (GHB), and diphosphoglycerate (DPG) and the number of white blood cells (WBCs) in the improved group (mice treated with modified preservative solution) significantly reduced compared with those in the ordinary group (Table 1, p < 0.05), indicating that the use of modified preservative solution led to better preservation of the physiological function of blood cells. Furthermore, qRT-PCR and western blot analysis were carried out in order to evaluate the effect of autologous blood preservatives on the expression of MALAT1 and HIF-1α. The results showed that the expression of MALAT1 and HIF-1α was significantly higher in the improved group (mice treated with modified preservative solution) than that in the ordinary group (Figures 1B and 1C, p < 0.05). The aforementioned results indicated that modified preservation of autologous blood elevated the expression of MALAT1 and further activated the HIF-1α signaling pathway, thus improving the biological and physiological function of blood cells.

Figure 1

Modified Autologous Blood Preservatives Increased the Expression of MALAT1 and Activated the HIF-1α Signaling Pathway

(A) Platelet aggregation rate determined by flow cytometry. (B) The mRNA expression of MALAT1 and HIF-1α in each group determined by qRT-PCR. (C) The protein expression of HIF-1α in each group normalized to GAPDH determined by western blot analysis. The results were measurement data and expressed as mean ± SD. Comparisons between two groups were analyzed using Student’s t test, whereas comparisons among multiple groups were assessed using ANOVA, followed by Tukey’s post hoc test. n = 10. The experiment was repeated three times independently.

Table 1

BG, GHB, and DPG Levels and the Number of WBCs Reduced in the Improved Group

Blood Physiological Indicator	Improved Group	Ordinary Group	p Value
BG (mmol/L)	7.59 ± 0.63	16.56 ± 1.23*	<0.001
GHB (mmol/L)	13.19 ± 0.97	24.45 ± 1.68*	<0.001
DPG (mmol/L)	1.78 ± 0.15	2.57 ± 0.21*	<0.001
WBCs (109/L)	6.85 ± 0.86	9.29 ± 1.03*	0.003

BG, blood glucose; DPG, diphosphoglycerate; GHB, glycosylated hemoglobin; WBC, white blood cell. The measurement data: Student’s t test was used for data analysis; the experiment was repeated three times; *p < 0.05 versus the ordinary group.

Modified Autologous Blood Preservatives Enhanced Fibroblast Activation of Diabetic Mice through MALAT1 Upregulation

The expression of MALAT1 and HIF-1α in the skin tissues of diabetic mice was verified to significantly increase when the blood was preserved in the modified preservative solution. qRT-PCR and western blot analysis were then carried out in order to determine the factors related to fibroblast activation (fibroblast activation protein [FAP], type I collagen [Col I], and Col III) so as to further elucidate the effects of MALAT1 on fibroblast activation, and the results are shown in Figures 2A and 2B. In comparison with the ordinary group, the expression of vascular endothelial growth factor-A (VEGF-A), matrix metalloproteinase-9 (MMP-9), FAP, Col I, and Col III was found to significantly increase in the improved group (mice treated with modified preservative solution) (all p < 0.05). The results showed that the modified preservation increased the expression of MALAT1 and activated the HIF-1α signaling pathway in mice undergoing blood transfusion, thereby enhancing the activation of fibroblasts.

Figure 2

The Modified Autologous Blood Preservatives Enhanced Fibroblast Activation by Upregulating the Expression of MALAT1

(A) The mRNA expressions of fibroblast activation-related genes determined by qRT-PCR. (B) The expression levels of proteins related to fibroblast activation normalized to GAPDH determined by western blot analysis. (C) qRT-PCR was performed to determine the expression of MALAT1. (D) The mRNA expression of HIF-1α, FAP, MMP-9, VEGF-A, Col I, and Col III after the overexpression of MALAT1 under the condition of HG determined by qRT-PCR. (E) The protein expressions of HIF-1α, FAP, MMP-9, VEGF-A, Col I, and Col III normalized to GAPDH after the overexpression of MALAT1 under the condition of HG determined by western blot analysis. (F) The viability of fibroblasts detected by CCK-8 assay. *p < 0.05 versus the ordinary, oe-MALAT1-NC (mouse fibroblasts cultured in the presence of oe-MALAT1-NC), or HG (mouse fibroblasts cultured in high-glucose medium) group. The results were measurement data and expressed as mean ± SD. Comparisons between two groups were analyzed using t test, whereas comparisons among multiple groups were assessed using ANOVA or repeated-measures ANOVA, followed by Tukey’s post hoc test. The experiment was repeated three times independently.

In addition, the fibroblasts were cultured under a high-glucose (HG) condition, and we also overexpressed MALAT1. qRT-PCR was carried out to determine the expression of MALAT1. As shown in Figure 2C, the relative expression of MALAT1 in the mice treated with oe-MALAT1 was significantly higher than that in the mice treated with oe-MALAT1 negative control (NC), indicating that MALAT1 overexpression was effective. qRT-PCR and western blot analysis were used to determine the expression of factors related to fibroblast activation. Results are shown in Figures 2D and 2E. Compared with the human overexpressed (H-oe)-MALAT1-NC group, the expressions of MALAT1, HIF-1α, FAP, MMP-9, VEGF-A, Col I, and Col III were found to significantly increase in fibroblasts of the H-oe-MALAT1 group (all p < 0.05), and there were no significant differences between the HG group and the H-oe-MALAT1-NC group (p > 0.05). In addition, the results of the Cell Counting Kit-8 (CCK-8) assay showed that, compared with the H-oe-MALAT1-NC group, the H-oe-MALAT1 group presented with significantly increased fibroblast viability (Figure 2F) (p < 0.05), and there were no significant differences between the HG group and the H-oe-MALAT1-NC group (p > 0.05). These results demonstrated that MALAT1 overexpression in fibroblasts cultured in HG conditions significantly increased the expression of fibroblast activation marker FAP and the expressions of Col I and Col III. Therefore, MALAT1 promoted the viability of fibroblasts and increased the activation of fibroblasts under HG media.

Modified Autologous Blood Preservatives Promoted Wound Healing in Diabetic Mice through MALAT1 Upregulation

The current study constructed a full-thickness skin wound model in order to assess the effect of MALAT1 on wound healing in diabetic mice. H&E staining was carried out for pathological observation on the wounded skin tissues of diabetic mice after autologous blood treated with modified preservative solution was transfused into mice. Subsequently, the wound healing rate was calculated and recorded. The results showed that, compared with the ordinary group, the wound healing rate in the improved group significantly increased on the 7th, 10th, and 14th days (Figure 3A, p < 0.05). As shown in Figure 3B, compared with the ordinary group, the number of inflammatory cells in the surface of the wound in the improved group was found to decrease, and the fibroblasts were found to increase. In order to further determine the expression of related indicators of wound healing, immunohistochemistry was used to measure the positive rates of Col I and Col III in the skin tissues. If the extracellular matrix showed a linear and reticular brown reaction, and if its staining intensity was higher than that of non-specific background staining, the corresponding regions were regarded to be positively stained. Compared with the ordinary group, the percentage of positive areas of Col I and Col III was found to significantly increase in the improved group (Figure 3C, p < 0.05). The above results suggested that the indicators related to wound healing were better expressed in the improved group than those in the ordinary group, and wound healing was faster in the improved group. Because these results illustrated that the expression of MALAT1 increased in the improved group, there may be a certain association between the expression of MALAT1 and wound recovery. In order to further verify whether that MALAT1 expression could promote wound recovery, the expression of wound healing-related indicators was measured in diabetic mice treated with short hairpin (sh)-MALAT1-NC and sh-MALAT1 after transfusion of modified autologous blood. The results demonstrated that the wound healing rate of diabetic mice treated with sh-MALAT1 was significantly lower than that in the mice treated with sh-MALAT1-NC on the 7th, 10th, and 14th days (Figure 3D, p < 0.05). H&E staining (Figure 3E) showed that, compared with the mice treated with sh-MALAT1-NC, the number of inflammatory cells in the surface of the wound in the mice treated with sh-MALAT1 was found to be larger and the number of fibroblasts was found to be smaller. Immunohistochemistry (Figure 3F) showed that, compared with the diabetic mice treated with sh-MALAT1-NC, the staining of Col I and Col III of the skin tissues in the diabetic mice treated with sh-MALAT1 was obviously lighter, and the percentage of positive areas of Col I and Col III decreased significantly (p < 0.05). The results indicated that MALAT1 silencing inhibited wound healing and reduced collagen expression in wounded areas.

Figure 3

Modified Autologous Blood Preservatives Promoted Wound Healing in Diabetic Mice by Upregulating the Expression of MALAT1

(A) The healing rate measured using a full-thickness skin wound model of diabetic mice. (B) The number of inflammatory cells and fibroblasts in the wound surface identified by H&E staining. (C) The percentage of positive areas of Col I and Col III in the tissues identified by immunochemistry (original magnification ×200). (D) The wound healing rate of diabetic mice treated with sh-MALAT1 using a full-thickness skin wound model in diabetic mice. (E) The number of inflammatory cells and fibroblasts in the diabetic mice treated with sh-MALAT1 identified by H&E staining (original magnification ×200). (F) The percentage of positive area of Col I and Col III in the diabetic mice treated with sh-MALAT1 identified by immunochemistry (original magnification ×200). The results were measurement data, expressed as mean ± SD and evaluated by Student’s t test. n = 10. The experiment was repeated three times independently.

MALAT1 Promoted Wound Healing in Diabetic Mice via Activating the HIF-1α Signaling Pathway

In order to verify the relationship between MALAT1 and the HIF-1α signaling pathway, the expression of MALAT1 was promoted and inhibited in fibroblasts, respectively. Then the results of qRT-PCR and western blot analysis showed that the expression of HIF-1α and VEGF-A in fibroblasts was significantly lower in the mice treated with sh-MALAT1 than that of the mice treated with sh-MALAT1-NC. Compared with the oe-MALAT1-NC group, the expression of HIF-1α and VEGF-A significantly increased in the oe-MALAT1 group (Figures 4A and 4B, both p < 0.05). The aforementioned results indicated that MALAT1 could activate the HIF-1α signaling pathway. Meanwhile, qRT-PCR and western blot analysis were used to examine the factors related to fibroblast activation. It was suggested that, compared with the mice treated with oe-MALAT1 + sh-HIF-1α NC, the expression of FAP, MMP-9, VEGF-A, Col I, and Col III significantly decreased in the mice treated with oe-MALAT1 + sh-HIF-1α (Figures 4C and 4D, all p < 0.05). CCK-8 assay was carried out to detect the cell proliferation ability, and it was suggested that, compared with the mice treated with oe-MALAT1 + sh-HIF-1α NC, the cell proliferation ability of the mice treated with oe-MALAT1 + sh-HIF-1α significantly reduced (Figure 4E, all p < 0.05). These results suggested that MALAT1 promoted fibroblast activity by activating the HIF-1α signaling pathway.

Figure 4

Modified Autologous Blood Preservatives Promoted Wound Healing of Diabetic Mice by Upregulating the Expression of MALAT1 and HIF-1α

(A) The mRNA expression of genes related to cell activation determined by qRT-PCR after overexpression and inhibition of MALAT1. (B) The protein expression of genes related to cell activation normalized to GAPDH determined by western blot analysis after overexpression and inhibition of MALAT1. (C) The mRNA expression of HIF-1α signaling pathway-related genes (HIF-1α, MMP-9, VEGF-A, FAP, Col I, and Col III) determined by qRT-PCR. (D) The protein expression of HIF-1α signaling pathway-related genes (HIF-1α, MMP-9, VEGF-A, FAP, Col I, and Col III) normalized to GAPDH determined by western blot analysis. (E) Fibroblast viability detected by CCK-8 assay. The results were measurement data, expressed as mean ± SD and evaluated by one-way ANOVA. The experiment was repeated three times independently.

Modified Autologous Blood Preservatives Promoted the Wound Healing of Diabetic Mice through MALAT1 Upregulation

In order to further explore the effect of HIF-1α on wound healing in diabetic mice, HIF-1α expression was interfered with in diabetic mice with improved blood transfusion, and a full-thickness skin wound model was established. It was found that the wound healing rate in mice treated with sh-HIF-1α decreased in comparison with the mice treated with sh-HIF-1α NC. Furthermore, H&E staining demonstrated that compared with the mice treated with sh-HIF-α NC, the number of inflammatory cells in the surface of the wound was found to be larger and the number of fibroblasts was found to be smaller in the mice treated with sh-HIF-1α (Figures 5A and 5B, p < 0.05). If extracellular matrix showed a linear and reticular brown reaction, and its staining intensity was higher than that of non-specific background staining, it was regarded as immunohistochemical positive staining. Compared with the mice treated with sh-HIF-α NC, the staining of Col I and Col III in the skin tissues of mice treated with sh-HIF-α was observed to be lighter. The percentage of Col I- and Col III-positive areas also decreased significantly in the mice treated with sh-HIF-α (Figure 5C, p < 0.05). It could be seen that inhibition of HIF-1α in the mice treated with the modified preservative solution suppressed the synthesis of collagen and the progress of wound healing. In addition, the activation of fibroblasts was compared between the two groups. The results of qRT-PCR and western blot analysis showed that the expression of HIF-1α, FAP, MMP-9, VEGF-A, Col I, and Col III in the mice treated with sh-HIF-α decreased significantly compared with those in the mice treated with sh-HIF-α NC. However, there were no significant differences between the two groups in terms of MALAT1 expression (Figures 5D and 5E, p > 0.05). Furthermore, CCK-8 assay was employed to detect the viability of fibroblasts. Compared with the mice treated with sh-HIF-α NC, the viability of fibroblasts in the mice treated with sh-HIF-α was found to significantly decrease (Figure 5F, p < 0.05). These results suggested that the modified preservative solution of autologous blood activated the HIF-1α signaling pathway by upregulating MALAT1, thus promoting fibroblast activation and wound healing in diabetic mice.

Figure 5

Modified Autologous Blood Preservatives Promoted Wound Healing of Diabetic Mice by Activating the HIF-1α Signaling Pathway

(A) The healing rate measured using a full-thickness skin wound model in diabetic mice (n = 10). (B) The number of inflammatory cells and fibroblasts identified by H&E staining (original magnification ×200) (n = 10). (C) The percentage of Col I- and Col III-positive area identified by immunohistochemistry (original magnification ×200). (D) The mRNA expression of HIF-1α, FAP, MMP-9, VEGF-A, Col I, and Col III determined by qRT-PCR. (E) The protein expression of HIF-1α, FAP, MMP-9, VEGF-A, Col I, and Col III normalized to GAPDH determined by western blot analysis. (F) Fibroblast proliferation detected by CCK-8 assay. The results were measurement data, expressed as mean ± SD and measured by Student’s t test. The experiment was repeated three times independently.

Discussion

As dynamic processes involving coagulation, inflammation, and fibroplasia, wound healing and wound infection are affected by obesity, macrovascular disease, and microvascular disease, especially in diabetic patients.16, 17, 18, 19, 20 However, the pathophysiologic relationship between diabetes and impaired healing remains to be determined. In the current study, we aimed to clarify whether MALAT1 could exert effects on wound healing in diabetic mice. Consequently, this study demonstrated that upregulated MALAT1 in the autologous blood of diabetic mice treated by modified preservatives could promote the activation of fibroblasts, thus accelerating wound healing of diabetic mice with the involvement of the HIF-1α signaling pathway.

First, the present study revealed that MALAT1 was upregulated in autologous blood samples of diabetic mice that had undergone treatment with modified blood preservatives. In the previous literature, autologous blood transfusion reduced the patients’ need for allogeneic transfusion after the resection of esophageal cancer, thus lowering the risk for postoperative infection. In addition, it was reported that autologous blood transfusion causes no damage to erythrocytes in diabetic patients undergoing off-pump coronary artery bypass grafting, thus further eliciting the beneficial use of autologous blood transfusion. Despite the substantial efficacy of autologous blood transfusion in diabetic patients, the current study proposed a modified blood preservative formula for autologous blood transfusion in order to prevent the accumulation of advanced glycation end products, to maintain the stability of the erythrocyte membrane, and to improve its oxygen-carrying ability. Accordingly, the diabetic mice treated with modified blood preservatives showed improved blood platelet aggregation.

During subsequent experimentation, we investigated the role of MALAT1 in the activation of fibroblasts and wound healing of diabetic mice. The results showed that the expressions of FAP, Col I, and Col III were significantly increased in the improved group, indicating that MALAT1 promoted the viability of fibroblasts and stimulated fibroblast activation. As a newly discovered lncRNA, MALAT1 was previously reported to be aberrantly expressed in a wide array of cancers, functioning as a novel potential biomarker for the prognosis of cancer. In addition, as a key regulatory factor in the pathogenesis of systemic lupus erythematosus, MALAT1 was reported to act as a novel target for therapeutic interventions. Similarly, Li et al. demonstrated that MALAT1 can regulate renal tubular epithelial pyroptosis in diabetic nephropathy. In addition, the overexpression of MALAT1 plays a pathogenic role in diabetes-induced microvascular dysfunction, further indicating the role played by MALAT1 in diabetes. Nevertheless, the role of MALAT1 in fibroblasts remains to be explored. Our results highlighted the therapeutic potential of MALAT1 in fibroblast activation by analyzing the expression patterns of factors related to fibroblast activation. Meanwhile, our findings also suggested the correlations between MALAT1 and wound healing in diabetic mice. Using a loss-of-function analysis, MALAT1 silencing was found to contribute to decreased expressions of Col I and Col III in skin tissue of diabetic mice, thereby reducing collagen deposition in wound areas and preventing wound healing.

In addition, we also explored the effect of HIF-1α on fibroblast activation and wound healing. Inhibition of HIF-1α was found to suppress the synthesis of collagen, inhibit fibroblast activation, and impair wound healing. HIF-1α was also reported to regulate lymphangiogenesis by mediating the expression of lymphangiogenic cytokines during wound healing. Another study also found that HIF-1α overexpression in diabetic mice promotes angiogenesis and helps the treatment of cutaneous wounds. Importantly, HIF-1α inhibits the fibroblast-like markers Col I and Col III in the re-differentiation of chondrocytes. Furthermore, the relationship between MALAT1 and HIF-1α was identified by a dual-luciferase report gene assay, and the results showed that MALAT1 targeted the HIF-1α promoter region, demonstrating direct correlations. Thus, MALAT1 positively regulates HIF-1α. Partially consistent with our results, a previous study found that MALAT1 increases arsenite-induced glycolysis in human hepatic epithelial cells by stabilizing the levels of HIF-1α. Based on these results, it can be inferred that MALAT1 promotes wound healing by activating the HIF-1α signaling pathway in diabetic mice.

In summary, our data supported the notion that, through the activation of the HIF-1α signaling pathway, upregulated MALAT1 enhances fibroblast activation, resulting in the accumulation of collagen deposition and enhancement of wound healing in diabetic mice treated with modified autologous blood preservatives. Therefore, the current study suggests that MALAT1 could serve as a potential therapeutic target in diabetic patients suffering from wound healing problems. Moreover, the improved group exhibited a significant improvement in BG reduction compared with the ordinary group. However, the influence of BG concentration on wound healing remains controversial, and the underlying mechanism warrants further investigation. Furthermore, future research on glycemic improvement is required to evaluate its potential in the treatment of impaired wound healing in diabetic patients.

Materials and Methods

Ethics Statement

All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the NIH and the approval of the Ethics Committee of Gongli Hospital, The Second Military Medical University. All efforts were made to minimize the suffering of the included animals.

Establishment of a Mouse Model of Diabetes

A total of 50 healthy Institute of Cancer Research Swiss male mice (clean-grade 6+, calculated mean weight of 23 ± 2 g) were obtained from Shanghai Slac Laboratory Animal (license number: SCXK [Hu] 2016-0005, Shanghai, China). These mice were raised in a mesh cage using a light-dark regimen comprising 12 h of illumination (6:00 a.m. to 6:00 p.m.) and 12 h of darkness (6:00 p.m. to 6:00 a.m.). The surrounding temperature was maintained at 25°C, and the mice were acclimated for 1 week prior to experimentation. A mouse model of diabetes was established by subcutaneous infusion of streptozotocin (STZ) (Sigma, St. Louis, MO, USA) every other day. If significantly elevated levels of blood sugar, daily urination, and daily water intake were found in experimental mice accompanied with significant weight loss within 2–4 weeks after STZ infusion (a total of five infusions), the diabetes mouse model was considered to be successfully established.32, 33 After establishment, diabetic mice were randomly divided into the two following groups: the improved group (the 30 mice in this group were transfused with the modified autologous blood) and the ordinary group (the 10 mice in this group were transfused with the ordinary autologous blood). At first, 0.5 mL of peripheral venous blood samples was obtained from each diabetic mouse. Subsequently, the blood samples obtained from the mice in the improved group were immediately placed in an improved preservative solution and preserved for 7 days for further experimentation. The improved preservative solution (pH 6.5) was composed of 2.0 mmol/L adenine, 55.5 mmol/L sugar, 55 mmol/L mannitol, 26 mmol/L sodium chloride, and 12 mmol/L sodium hydrogen phosphate, with an osmotic pressure of 286 mOsm/kg (Advanced Fiske210; Advanced Instruments, Norwood, MA, USA). Similarly, the blood samples obtained from the mice in the ordinary group were stored in an ordinary preservative solution before further experimentation. The ordinary preservative solution (pH 5.8) was composed of 2.2 mmol/L adenine, 110 mmol/L sugar, 70.1 mmol/L sodium chloride, 20 mmol/L sodium hydrogen phosphate, and 12 mmol/L citric acid, with an osmotic pressure of 264 mOsm/kg. Compared with the ordinary preservative solution, the modified preservative solution contained mannitol that could stabilize the permeation of RBCs, and sugar levels were reduced to prevent the accumulation of advanced glycosylation terminal products on erythrocyte membrane so as to maintain the stability of the erythrocyte membrane and improve its oxygen-carrying function in diabetic mice. Blood physiological tests were carried out 7 days later. All mice were raised in individual cages for subsequent experiments and were allowed free access to water and food during the experiment.

Flow Cytometry

After collection, peripheral venous blood samples from the two groups were preserved for 7 days, and the platelet aggregation rate was detected using a double-staining method with fluorescein isothiocyanate (FITC) and phycoerythrin (PE). The preserved blood was treated with 0.01 M PBS and platelet-rich plasma (PRP) to allow platelet adhesion, followed by fluorescence labeling of platelets. The mixture of CD41-PE and CD41-FITC was supplemented with 0.8 μL of ADP (at a final concentration of 20 μM) and vortexed for 5 min (800 rpm) for platelet activation without light exposure. Meanwhile, the mixture of CD41-PE and CD41-FITC was supplemented with 0.8 μL of 0.01 M PBS and vortexed for 5 min (800 rpm), serving as the NC. The reaction was then terminated with the addition of 1 mL of a 1% pre-cooled (2°C–8°C) polyformaldehyde PBS. The mixture was then transferred into a flow cytometer, and flow-count fluorescent microspheres were added for 30 min of incubation at 4°C to assess the platelet aggregation rate. The proportion of CD41-PE- and CD41-FITC-positive platelets in the total platelets was calculated as the platelet aggregation rate. The fluorescence signals on the surface of 5,000–10,000 platelets were detected in each specimen.

Examination of Blood Physiological Indicators

Venous blood specimens from mice in the two groups were stored in different preservative solutions for 7 days. A total of five autologous blood specimens from the mice in each group were added to EDTA anticoagulant tubes. The levels of BG, GHB, and DPG were measured, and the number of WBCs was counted using an SYSMEX XE-2100 automated hematology analyzer.

qRT-PCR

Total RNA from the skin tissues of mice from the two groups was extracted using the TRIzol one-step method using a TRIzol kit (15596-026; Invitrogen, Gaithersburg, MD, USA). RNA concentration was measured using nucleic acid-protein analyzer (BioPhotometer D30; Eppendorf, Hamburg, Germany). Subsequently, total RNA was reversely transcribed into cDNA based on the instructions of a cDNA Synthesis kit (K1621; Fermentas, Maryland, NY, USA). The obtained cDNA was then preserved for further use in a freezer at −20°C. The primer sequences (Table 2) were designed and synthesized by Shanghai Genechem (Shanghai, China). The mRNA expression of each gene was determined using a fluorescent qPCR Kit (Takara, Dalian, Liaoning, China) in a real-time fluorescent qPCR instrument (ABI 7500; ABI, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal reference.

Table 2

Primer Sequences for qRT-PCR

Gene	Primer Sequences
lncRNA MALAT1	F: 5′-CTAGTTTGAAGGTCGGCCT-3′
	R: 5′-TCTCCAAATACTAGCCTAACCTC-3′
HIF-1α	F: 5′-GCACGTCATGGGTGGTTTCT-3′
	R: 5′-GGGAGGACGATGAACATCAA-3′
VEGF-A	F: 5′-CCATGAACTTTCTGCTCTTC-3′
	R: 5′-GGTGAGAGGTCTAGTTCCCGA-3′
FAP	F: 5′-GCACTTTCAGAACTCAGCA-3′
	R: 5′-TGGTCAGAGTACCACATCG-3′
MMP-9	F: 5′-GCAGAGGCATACTTGTACCG-3′
	R: 5′-TGATGTTATGATGGTCCCACTTG-3′
Col I	F: 5′-CATGTTCAGCTTGTGGACCT-3′
	R: 5′-GCAGCTGACTTCAGGGATGT-3′
Col III	F: 5′-TCCCCTGGAATCTGTGAATC-3′
	R: 5′-TGAGTCGAATTGGGGAGAAT-3′
GAPDH	F: 5′-AGGTCGGTGTGAACGGATTTG-3′
	R: 5′-GGGGTCGTTGATGGCAACA-3′

Col I, type I collagen; Col III, type III collagen; F, forward; FAP, fibroblast activation protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIF-1α, hypoxia-inducible factor-1α; lncRNA, long non-coding RNA; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; MMP-9, matrix metalloproteinase-9; R, reverse; VEGF-A, vascular endothelial growth factor-A.

Western Blot Analysis

A protein lysis buffer (R0010; Beijing Solarbio Science and Technology, Beijing, China) containing a protease inhibitor cocktail was added to the skin tissue samples of mice from the two groups. The skin tissues were then homogenized at 3,000 rpm for thorough lysis, followed by incubation in an ice bath for 30 min, and then centrifuged at 1,2000 rpm for 15 min at 4°C to collect the supernatant. The protein concentration was determined using a bicinchoninic acid (BCA) kit (23225; Pierce, Rockford, IL, USA), and the concentration of protein was adjusted to 1 μg/μL. The protein was then separated by 10% SDS-PAGE (P1200; Beijing Solarbio Science and Technology, Beijing, China) using 20 μg/well of the sample. The separated protein samples were then transferred onto polyvinylidene fluoride membranes (HVLP04700; Millipore, Bedford, MA, USA) using the semidry-electrophoretic transfer method. Subsequently, the membranes were rinsed two times using Tris-buffered saline with Tween (TBST), blocked with 5% skim milk at room temperature for 2 h, rinsed again with TBST three times, and incubated in a refrigerator at 4°C overnight with primary mouse antibodies against HIF-1α (dilution ratio of 1:1,000, ab16066), VEGF-A (dilution ratio of 1:1,500, ab38909), MMP-9 (dilution ratio of 1:1,000, ab58803), Col I (dilution ratio of 1:1,000, ab90395), Col III (dilution ratio of 1:5,000, ab23445), and β-actin (dilution ratio of 1:10,000, ab8226) (all obtained from Abcam, Cambridge, MA, USA). After the membranes were rinsed with TBST for a total of three times (10 min each time), they were incubated with horseradish peroxidase (HRP)-labeled secondary antibody of goat anti-mouse antibody to immunoglobulin G (IgG) (dilution ratio of 1:2,000, ab6721; Abcam, Cambridge, MA, USA) at room temperature for 2 h. Following three TBST rinses (10 min each time), the membranes were stained using diaminobenzidine (DAB). The protein bands were visualized in a Molecular Imager Gel Doc XR system (Bio-Rad, Hercules, CA, USA). The ratio of the gray value of a target protein to that of the internal reference was calculated as the relative expression of the target protein. This method was also suitable for the detection of cellular protein levels.

Isolation and Culture of Mouse Fibroblasts

The week following the successful modeling, five mice were euthanized, soaked in 75% alcohol for 5 min, and rinsed with PBS three times. The mice were then fixed to remove the hair and to clean the skin. Subsequently, the extracted skin of the tail, back, and ear was placed in a Petri dish containing PBS. Irreverent tissues, such as fat and blood vessels, were carefully removed, and the remaining tissues were rinsed with PBS three times, followed by 2 h of detachment using 2 mL trypsin. Subsequently, the epidermis was removed after the separation of corium and epidermis, and the corium was rinsed with PBS three times. About 1-cm3 tissues were extracted and washed with D-Hank’s solution, and the supernatant was discarded. The tissues were cultured in a low-sugar DMEM containing 0.15% collagenase. The mixture was then added to aseptic centrifuge tubes, treated on the electromagnetic agitator at 37°C, and centrifuged at 700 rpm for 5 min. The supernatant was discarded, and a suitable amount of low-sugar DMEM containing 20% fetal bovine serum (FBS) was added. The mixture was centrifuged at 700 rpm again, and the precipitate was added to the medium in order to prepare a cell suspension. After uniform mixing, the mixture was transferred into a disposable Petri dish, so that the precipitate was evenly distributed in the culture dish. After being washed with D-Hank’s solution, the mixture was detached with 0.25% trypsin (containing 0.02% EDTA) at 37°C in a 5% CO2 incubator for 7–9 min, and a medium containing 20% FBS was added to terminate the detachment. The mixture was centrifuged again at 1,200 rpm for 5 min, and the supernatant was discarded. The obtained fibroblasts were re-suspended in a DMEM containing 20% FBS, inoculated into a new culture dish, and cultured in a 5% CO2 incubator at 37°C.

Construction of Lentiviral Vectors

Lentiviral interference vectors, pSIH1-H1-copGFP (sh-, interference vector), and lentiviral overexpression vector, pLV-EGFP-N (oe-, overexpression vector), were purchased from Shanghai GenePharma (Shanghai, China). A lentiviral-based MALAT1 interference vector, a MALAT1 overexpression vector, and a HIF-1α gene silencing vector were constructed.

Cell Transfection and Grouping

The fibroblasts at passage 3 were selected and divided into different groups as shown in Table 3. A total of 100 pmoL lentiviral vector from each group was diluted with 250 μL of serum-free Opti-MEM (GIBCO, Grand Island, NY, USA) to a final concentration of 50 nM and then added to a six-well cell culture plate. After transfection, the lentiviral vector was then incubated at 37°C with 5% CO2 in saturated humidity. After incubation for 24 h, the medium containing the transfection solution in the well was discarded and replaced with RPMI 1640 medium containing 10% FBS (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Incubation was continued for 24–48 h for subsequent experiments.

Table 3

Cell Transfection and Grouping

Group	Lentivirus	Medium
H-oe-MALAT1	oe-MALAT	5.5 mmol/L glucose
H-oe-MALAT1-NC	oe-MALAT-NC	5.5 mmol/L glucose
oe-MALAT1	oe-MALAT	conventional
sh-MALAT1	sh-MALAT	conventional
oe-MALAT1-NC	oe-MALAT-NC	conventional
sh-MALAT1-NC	sh-MALAT-NC	conventional
oe-MALAT1 + sh-HIF-1α NC	oe-MALAT + sh-HIF-1α NC	conventional
oe-MALAT1 + sh-HIF-1α	oe-MALAT + sh-HIF-1α	conventional

H-oe, human overexpressed; HIF-1α, hypoxia-inducible factor-1α; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; NC, negative control.

CCK-8 Assay

The viability of fibroblasts was assessed using a CCK-8 kit (GM-040101-5; Dojindo, Gaithersburg, MD, USA). The transfected fibroblasts at the logarithmic phase of growth were collected and cultured in 96-well plates at a concentration of 5 × 103 cells/μL. Cell viability was assessed using 10 μL of CCK-8 reagent after every 24 h of cell culture. The fibroblasts were cultured for 4 h each time, and the optical density value at 450 nm was measured using a microplate reader.

Grouping of Diabetic Mice with Autologous Blood Re-transfusion

The previously established improved model mice were randomly selected and further divided into the improve (the mice in the improve group were injected with autologous blood preserved by modified solution via tail vein), sh-MALAT1-NC (the mice in the improve group were injected with autologous blood preserved by modified solution and sh-MALAT1-NC lentivirus via tail vein), sh-MALAT1 (the mice in the improve group were injected with autologous blood preserved by modified solution and sh-MALAT1 lentivirus via tail vein), sh-HIF-1α (the mice in the improve group were injected with autologous blood preserved by modified solution and sh-HIF-1α lentivirus via tail vein), and sh-HIF-1α NC (the mice in the improve group were injected with autologous blood preserved by modified solution and sh-HIF-1α NC lentivirus via tail vein) groups (five mice in each group). The mice were raised in individual cages with free access to water and food.

A Full-Thickness Skin Wound Model of Diabetic Mice

Mice from each group were anesthetized with 1% pentobarbital sodium, the skin overlying the back was disinfected, and full-thickness skin wounds of 1 cm in diameter were made on both sides of the spine with a self-made perforator. The area of the wound was recorded using a hyaline membrane tracing method for 7, 10, and 14 days post-injury. The collected data were processed by Image-Pro Plus software (Media Cybernetics, MD, USA) in conjunction with Excel 2000. The percentage of wound healing area was calculated according to the following formula: (initial area − residual area)/initial area × 100%.

H&E Staining

Fourteen days after the full-thickness skin wound model was established, wound tissues were extracted and fixed in 10% neutral formaldehyde solution for over 24 h. The specimens were dehydrated with conventional gradient alcohol (ethanol concentrations of 70%, 80%, 90%, 95%, 100%) for 1 min per time, cleared with xylene two times (5 min per time), embedded with paraffin, and sliced into 4-μm sections (some sections were used for immunohistochemistry). Then, the slices were baked in an oven at 80°C for 1 h, dehydrated with conventional gradient alcohol, and cleared with xylene. Subsequently, the sections were stained with hematoxylin (H8070-5g; Beijing Solarbio Technology, Beijing, China) for 4 min and then washed with water and differentiated by hydrochloric acid alcohol for 10 s. The slices were then rinsed, immersed in water for 5 min, treated with a 1% ammonium hydroxide solution for 10 min, stained with eosin (PT001; Shanghai Bogoo Biological Technology, Shanghai, China) for 2 min, dehydrated with gradient alcohol (1 min each time), and cleared with xylene two times (1 min each time). The operation was conducted in a fume hood. The pathological changes in the samples were observed under an optical microscope (DMM-300D; Shanghai Cai Kang Optical Instrument, Shanghai, China) after neutral balsam mounting.

Immunohistochemistry

Fourteen days after damage models were established, the wounded tissues were collected, and the accumulation of Col I and Col III was detected using immunohistochemistry. The paraffin-embedded slices were baked in an oven at 60°C overnight, dewaxed with xylene, and dehydrated with gradient alcohol (100%, 95%, 80%, 70%, 5 min each). The slices were then washed under tap water for 5 min, and then rinsed with PBS three times (3 min each time). The slices were then subjected to microwave antigen retrieval with a 0.01 M citric acid buffer for 10 min and were allowed to cool down to room temperature. Subsequently, the slices were rinsed in PBS three times (3 min each time) and then soaked in a 0.3% H2O2-methanol solution for 20 min to eliminate endogenous peroxidase activity. After rinsed with PBS three times, the slices were blocked with 10% goat serum (36119ES03; Shanghai Yeasen Biotechnology, Shanghai, China) at room temperature for 10 min and treated with 1 μg/mL Col I antibody (ab139645; Abcam, Cambridge, MA, USA) and Col III antibody (ab139645; Abcam, Cambridge, MA, USA) at 4°C overnight. Instead of the primary antibody, PBS served as the NC. HRP-labeled IgG secondary antibody (dilution ratio of 1:1,000, ab6721; Abcam, MA, USA) was then added to the slides and incubated at room temperature for 30 min. The tissues were then stained with DAB (P0203; Beyotime Biotechnology, Nanjing, China) for 5 min, and the degree of coloration was controlled under a microscope. Finally, these slices were washed under tap water for 5 min, stained with hematoxylin for 3 min, differentiated with 1% hydrochloric acid for 5 s, and soaked in tap water for 10 min, followed by neutral balsam mounting. Subsequently, the slices were observed under a microscope and photographed. If the extracellular matrix showed a linear and reticular brown reaction, and if its staining intensity was higher than that of non-specific background staining, the corresponding area was regarded as positively stained. Three slices were selected from each sample, and five fields were selected from each slice. The ratio of the positively stained area (the ratio of positive staining area to the total area in a field of vision) was calculated using an MIG-2000 comprehensive image analysis system, and the mean value was obtained.

Statistical Analysis

Statistical analyses were performed using the Statistical Package of the Social Sciences (SPSS) software, version 21.0 (IBM, Armonk, NY, USA). Measurement data with normal distribution were expressed as mean ± SD. Comparisons among multiple groups were assessed using one-way ANOVA, whereas comparisons between two groups were analyzed using an independent t test. Data at different time points were compared by repeated-measures ANOVA, followed by Tukey’s post hoc test. The enumeration data were represented by percentage. A p value <0.05 was considered to be statistically significant.

Author Contributions

X.-Q.L., L.-S.D., J.-R.G., and X.-J.J. designed the study. Y.-Q.C., N.-N.Z., X.Z., H.-W.W., and L.Y. collated the data, carried out data analyses, and produced the initial draft of the manuscript. X.-Q.L. and L.-S.D. contributed to drafting the manuscript. All authors participated in the revised manuscript and have read and approved the final submitted manuscript.

Conflicts of Interest

The authors declare no competing interests.