The secretory leukocyte protease inhibitor (SLPI) in pathophysiology of non-communicable diseases: Evidence from experimental studies to clinical applications.

Non-communicable diseases (NCDs) are a worldwide health issue because of their prevalence, negative impacts on human welfare, and economic costs. Protease enzymes play important roles in viral and NCD diseases. Slowing disease progression by inhibiting proteases using small-molecule inhibitors or endogenous inhibitory peptides appears to be crucial. Secretory leukocyte protease inhibitor (SLPI), an inflammatory serine protease inhibitor, maintains protease/antiprotease balance. SLPI is produced by host defense effector cells during inflammation to prevent proteolytic enzyme-induced tissue damage. The etiology of noncommunicable illnesses is linked to SLPI's immunomodulatory and tissue regeneration roles. Disease phases are associated with SLPI levels and activity changes in regional tissue and circulation. SLPI has been extensively evaluated in inflammation, but rarely in NCDs. Unfortunately, the thorough evaluation of SLPI's pathophysiological functions in NCDs in multiple research models has not been published elsewhere. In this review, data from PubMed from 2014 to 2023 was collected, analysed, and categorized into in vitro, in vivo, and clinical studies. According to the review, serine protease inhibitor (SLPI) activity control is linked to non-communicable diseases (NCDs) and other illnesses. Overexpression of the SLPI gene and protein may be a viable diagnostic and therapeutic target for non-communicable diseases (NCDs). SLPI is also cytoprotective, making it a unique treatment. These findings suggest that future research should focus on these pathways using advanced methods, reliable biomarkers, and therapy approaches to assess susceptibility and illness progression. Implications from this review will help pave the way for a new therapeutic target and diagnosis marker for non-communicable diseases.

Recombinant human secretory leukocyte protease inhibitor (rhSLPI) coated titanium enhanced human osteoblast adhesion and differentiation.

Osseointegration is vital to success in orthopedic and dental reconstructions with implanted materials. The bone matrix or cells-particularly osteoblasts-are required to achieve functional contact on the implant surface. Osteoblast induction is therefore essential for osteogenesis to occur. Enhancement of osteoblast adhesion, proliferation, and differentiation, particularly by implant surface modifications, have been found challenging to develop. Secretory Leukocyte Protease Inhibitor (SLPI), a cation ionic protein with anti-inflammatory and anti-bacterial activities, showed activation in osteoblast proliferation and differentiation. However, the effects of coating recombinant human (rh) SLPI on a titanium alloy surface on human osteoblast adhesion, proliferation, and differentiation has never been investigated. In this study, titanium alloys (Ti-6Al-4V) were coated with rhSLPI, while human osteoblast adhesion, proliferation, differentiation, actin cytoskeletal organization, and gene expressions involved in cell adhesion and differentiation were investigated. The results indicate that coating titanium with 10-100 µg/ml rhSLPI enhanced the physical properties of the Ti surface and enhanced human osteoblast (hFOB 1.19) cell adhesion, activated actin dynamic, enhanced adhesive forces, upregulated integrins α1, α2, and α5, enhanced cell proliferation, mineralization, alkaline phosphatase activity, and upregulated ALP, OCN, and Runx2. This is the first study to demonstrate that coating SLPI on titanium surfaces enhances osseointegration and could be a candidate molecule for surface modification in medical implants.

Gelatin-coated silicon oxide nanoparticles encapsulated recombinant human secretory leukocyte protease inhibitor (rhSLPI) reduced cardiac cell death against an in vitro simulated ischaemia/reperfusion injury.

Ischemic Heart Disease (IHD) is the main global cause of death. Previous studies indicated that recombinant human secretory leukocyte protease inhibitor (rhSLPI) exhibits a cardioprotective effect against myocardial ischaemia/reperfusion (I/R) injury. However, SLPI has a short half-life in vivo due to digestion by protease enzymes in circulation. The application of nanoparticle encapsulation could be beneficial for SLPI delivery. Several types of nanoparticles have been developed to encapsulate SLPI and applied in some disease models. However, silica nanoparticles for rhSLPI delivery, particularly on myocardial I/R injury, have never been studied. In this study, we aimed to fabricate gelatin-covered silica nanoparticles (GSNPs) to encapsulate rhSLPI and cardioprotective effect of GSNP-SLPI against an in vitro simulated ischaemia/reperfusion (sI/R). Silica dioxide nanoparticles (SNPs) were fabricated followed by incubation with 0.33 mg/mL of rhSLPI. Then, SNPs containing rhSLPI were coated with gelatin (GSNPs). The GSNPs and rhSLPI-GSNPs were characterized by particle size, zeta potential, and morphology scanning electron microscope (SEM). The concentration of rhSLPI in rhSLPI-GSNPs and drug release was determined by ELISA. Then, cytotoxicity and cardioprotective effect were determined by incubation of GSNPs or rhSLPI-GSNPs with rat cardiac myoblast cell line (H9c2) subjected to simulated ischaemia/reperfusion (sI/R). The results showed the particle size of SNPs, GSNPs, and rhSLPI-GSNPs was 273, 300, and 301 nm, with a zeta potential of -57.21, -22.40, and -24.50 mV, respectively. One milligram of rhSLPI-GSNPs contains 235 ng of rhSLPI. The rhSLPI-GSNPs showed no cytotoxicity on cardiac cells. Treatment with 10 µg/ml of rhSLPI-GSNPs could significantly reduce sI/R induced cardiac cell injury and death. In conclusion, this is the first study to show successful of fabricating novel rhSLPI-encapsulating gelatin-covered silica nanoparticles (rhSLPI-GSNPs) and the cardioprotective effects of rhSLPI-GSNPs against cardiac cell injury and death from myocardial ischaemia/reperfusion.

Adeno-associated virus 9 vector-mediated cardiac-selective expression of human secretory leukocyte protease inhibitor attenuates myocardial ischemia/reperfusion injury.

Protease enzymes contribute to the initiation of cardiac remodeling and heart failure after myocardial ischemic/reperfusion (I/R) injury. Protease inhibitors attenuate protease activity and limit left ventricular dysfunction and remodeling. Previous studies showed the cardioprotective effect of secretory leukocyte protease inhibitor (SLPI) against I/R injury. However, overexpression of SLPI gene in cardiovascular diseases has only been investigated in an in vitro experiment. Here, cardiac-selective expression of the human secretory leukocyte protease inhibitor (hSLPI) gene and its effect on I/R injury were investigated. Adeno-associated virus (AAV) serotype 9 carrying hSLPI under the control of cardiac-selective expression promoter (cardiac troponin, cTn) was intravenously administered to Sprague-Dawley rats for 4 weeks prior to coronary artery ligation. The results showed that myocardial-selective expression of hSLPI significantly reduced infarct size, cardiac troponin I (cTnI), creatine kinase-MB (CK-MB), and myoglobin levels that all served to improve cardiac function. Moreover, overexpression of hSLPI showed a reduction in inflammatory cytokines, oxidatively modified protein carbonyl (PC) content, ischemia-modified albumin (IMA), and necrosis and cardiac tissue degeneration. In conclusion, this is the first study to demonstrate cardiac-selective gene delivery of hSLPI providing cardioprotection against myocardial I/R injury in an in vivo model.

Anti-Protease Activity Deficient Secretory Leukocyte Protease Inhibitor (SLPI) Exerts Cardioprotective Effect against Myocardial Ischaemia/Reperfusion.

Inhibition of proteases shows therapeutic potential. Our previous studies demonstrated the cardioprotection by the Secretory Leukocyte Protease Inhibitor (SLPI) against myocardial ischaemia/reperfusion (I/R) injury. However, it is unclear whether the cardioprotective effect of SLPI seen in our previous works is due to the inhibition of protease enzymes. Several studies demonstrate that the anti-protease independent activity of SLPI could provide therapeutic benefits. Here, we show for the first time that recombinant protein of anti-protease deficient mutant SLPI (L72K, M73G, L74G) (mt-SLPI) could significantly reduce cell death and intracellular reactive oxygen species (ROS) production against an in vitro simulated I/R injury. Furthermore, post-ischaemic treatment of mt-SLPI is found to significantly reduce infarct size and cardiac biomarkers lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) activity, improve cardiac functions, attenuate I/R induced-p38 MAPK phosphorylation, and reduce apoptotic regulatory protein levels, including Bax, cleaved-Caspase-3 and total Capase-8, in rats subjected to an in vivo I/R injury. Additionally, the beneficial effect of mt-SLPI was not significantly different from the wildtype (wt-SLPI). In summary, SLPI could provide cardioprotection without anti-protease activity, which could be more clinically beneficial in terms of providing cardioprotection without interfering with basal serine protease activity.

Anti-ischemic effect of Tamarindus indica L. seed extract against myocardial hypoxic injury.

Background: Ischemic heart disease is a leading cause of death in patients with cardiovascular disease. Natural products containing high antioxidant activity have been used as an alternative therapy to improve the living conditions of patients. In this study, we examine the protective effect of tamarind seed (TS) on myocardial hypoxic injury. Methods: The hypoxia model was mimicked by mineral oil overlayed on H9c2 cardiomyoblasts for 4 h. TS extract was pretreated and administered during the hypoxic condition. Radical scavenging activity of TS extract was measured and exhibited very potent antioxidant activities on 2,2-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. Results: TS extract at a concentration of 10 µg/ml significantly reversed the effect of hypoxia-induced cell death and intracellular reactive oxygen species (ROS) production. We also observed hypoxia-induced over-expression of both inflammatory cytokine mRNA and activation of cellular apoptosis. Pretreatment of TS extract significantly reduced hypoxia-induced HIF-1a and pro-inflammatory cytokine production, IL-1b and IL-6. The Western blot analysis for apoptotic regulatory molecules, caspase 3, caspase 8 and Bax proteins, also showed hypoxia injury reversal by TS extract treatment. Conclusions: The results suggest that the anti-ischemic effect of TS extract protects against hypoxia-induced injury and has potential to be an effective alternative therapy for ischemic heart disease and oxidative-damage related disease.

p38 MAPK Inhibitor (SB203580) and Metformin Reduces Aortic Protein Carbonyl and Inflammation in Non-obese Type 2 Diabetic Rats.

Microvascular and macrovascular diseases are the main causes of morbidity in type 2 diabetes patients through chronic hyperglycaemic condition via oxidative stress and inflammation. Reactive oxygen species (ROS) activate p38 MAPK phosphorylation and inflammation which enhances protein modification by carbonylation. The use of metformin and a p38 MAPK inhibitor is hypothesised to reduce ROS production and inflammation but effects of metformin and p38 MAPK inhibitor (SB203580) on ROS production and inflammation in vascular type 2 diabetes mellitus non-obese (T2DM) have not been investigated. The Goto-Kakizaki rat T2DM model was divided into three groups as T2DM, T2DM treated with 15 mg/kg bw metformin and T2DM treated with 2 mg/kg bw SB203580 for 4 weeks. Rat aortas were isolated and protein carbonyl (PC) contents were measured by spectrophotometric DNPH assay. Aortic IL-1ß level was determined by ELISA. Results showed that aortic PC contents in the T2DM group were significantly higher than in non-diabetic rats. Treatment with metformin or SB203580 significantly reduced PC contents while only metformin significantly reduced IL-1ß levels. Findings indicated that metformin reduced ROS production and inflammation in diabetic vessels and possibly reduce vascular complications in non-obese T2DM.

Post-Ischemic Treatment of Recombinant Human Secretory Leukocyte Protease Inhibitor (rhSLPI) Reduced Myocardial Ischemia/Reperfusion Injury.

Myocardial ischemia/reperfusion (I/R) injury is a major cause of mortality and morbidity worldwide. Among factors contributing to I/R injury, proteolytic enzymes could also cause cellular injury, expand the injured area and induce inflammation, which then lead to cardiac dysfunction. Therefore, protease inhibition seems to provide therapeutic benefits. Previous studies showed the cardioprotective effect of secretory leukocyte protease inhibitor (SLPI) against myocardial I/R injury. However, the effect of a post-ischemic treatment with SLPI in an in vivo I/R model has never been investigated. In the present study, recombinant human (rh) SLPI (rhSLPI) was systemically injected during coronary artery occlusion or at the onset of reperfusion. The results show that post-ischemic treatment with rhSLPI could significantly reduce infarct size, Lactate Dehydrogenase (LDH) and Creatine kinase-MB (CK-MB) activity, inflammatory cytokines and protein carbonyl levels, as well as improving cardiac function. The cardioprotective effect of rhSLPI is associated with the attenuation of p38 MAPK phosphorylation, Bax, caspase-3 and -8 protein levels and enhancement of pro-survival kinase Akt and ERK1/2 phosphorylation. In summary, this is the first report showing the cardioprotective effects against myocardial I/R injury of post-ischemic treatments with rhSLPI in vivo. Thus, these results suggest that SLPI could be used as a novel therapeutic strategy to reduce myocardial I/R injury.

Combination of metformin and p38 MAPK inhibitor, SB203580, reduced myocardial ischemia/reperfusion injury in non-obese type 2 diabetic Goto-Kakizaki rats.

Diabetic cardiomyopathy, especially myocardial ischemia reperfusion (I/R) injury, is a major cause of morbidity and mortality in type 2 diabetic patients. The increasing of basal p38 MAP Kinase (p38 MAPK) activation is a major factor that aggravates cardiac death on diabetic cardiomyopathy. In addition, metformin also shows cardio-protective effects on myocardial ischemia/reperfusion injury. In this study, we investigated the effect of the combination between metformin and p38 MAPK inhibitor (SB203580) in diabetic rats subjected to I/R injury. H9c2 cells were induced into a hyperglycemic condition and treated with metformin, SB203580 or the combination of metformin and SB203580. In addition, cells in both the presence and absence of drug treatment were subjected to simulated ischemia/reperfusion injury. Cell viability and cellular reactive oxygen species (ROS) were determined. Moreover, the Goto-Kakizaki (GK) rats were treated with metformin, SB203580, and the combination of metformin and SB203580 for 4 weeks. Diabetic parameters and cardiac functions were assessed. Finally, rat hearts were induced ischemia/reperfusion injury for the purpose of infarct size analysis and determination of signal transduction. A high-glucose condition did not reduce cell viability but significantly increased ROS production and significantly decreased cell viability after induced sI/R. Treatment using drugs was shown to reduce ROS generation and cardiac cell death. The GK rats displayed diabetic phenotype by increasing diabetic parameters and these parameters were significantly decreased when treated with drugs. Treatment with metformin or SB203580 could significantly reduce the infarct size. Interestingly, the combination of metformin and SB203580 could enhance cardio-protective ability. Myocardial I/R injury significantly increased p38 MAPK phosphorylation, Bax/Bcl-2 ratio and caspase-3 level. Treatment with drugs significantly decreased the p38 MAPK phosphorylation, Bax/Bcl-2 ratio, caspase-3 level and increased Akt phosphorylation. In conclusion, using the combination of metformin and SB203580 shows positive cardio-protective effects on diabetic ischemic cardiomyopathy.