Therapeutic potential of proteases in acute lung injury and respiratory distress syndrome via TLR4/Nrf2/NF-kB signaling modulation.

The COVID-19 pandemic has drawn attention to acute lung injury and respiratory distress syndrome as major causes of death, underscoring the urgent need for effective treatments. Protease enzymes possess a wide range of beneficial effects, including antioxidant, anti-inflammatory, antifibrotic, and fibrinolytic effects. This study aimed to evaluate the potential therapeutic effects of bacterial protease and chymotrypsin in rats in mitigating acute lung injury induced by lipopolysaccharide. Molecular docking was employed to investigate the inhibitory effect of bacterial protease and chymotrypsin on TLR-4, the receptor for lipopolysaccharide. Bacterial protease restored TLR-4, Nrf2, p38 MAPK, NF-kB, and IKK-ß levels to normal levels, while chymotrypsin normalized TLR-4, IKK-ß, IL-6, and IL-17 levels. The expression of TGF-ß, caspase-3, and VEGF in the bacterial protease- and chymotrypsin-treated groups was markedly reduced. Our results suggest that both therapies ameliorate LPS-induced acute lung injury and modulate the TLR4/Nrf2/NF-k signaling pathway. Each protease exhibited distinct mechanisms, with bacterial protease showing a better response to oxidative stress, edema, and fibrosis, whereas chymotrypsin provided a better response in the acute phase and innate immunity. These findings highlight the potential of each protease as a promising therapeutic option for acute lung injury and respiratory distress syndrome.

Bacterial protease alleviate chronic liver fibrosis induced by thioacetamide through suppression of hepatic stellate cells consequently decrease its proliferative index.

In chronic liver diseases, liver fibrosis occurs due to excessive extracellular matrix (ECM) protein accumulation. Approximately 2 million deaths occur yearly due to liver disease, while cirrhosis is the 11th most common cause of death. Therefore, newer compounds or biomolecules must be synthesized to treat chronic liver diseases. In this aspect, the present study focuses on the assessment of the anti-inflammatory and antioxidant impact of Bacterial Protease (BP) produced by a new mutant strain of bacteria (Bacillus cereus S6-3/UM90) and 4,4'-(2,5-dimethoxy-1,4-phenylene) bis (1-(3-ethoxy phenyl)-1H-1,2,3-triazole) (DPET) in the treatment of early stage of liver fibrosis induced by thioacetamide (TAA). Sixty male rats were divided into six groups, ten rats each as follows: (1) Control group, (2) BP group, (3) TAA group, (4) TAA-Silymarin (S) group, (5) TAA-BP group, and (6) TAA-DPET group. Liver fibrosis significantly elevated liver function ALT, AST, and ALP, as well as anti-inflammatory interleukin 6 (IL-6) and VEGF. The oxidative stress parameters (MDA, SOD, and NO) were significantly increased with a marked reduction in GSH. Expression of MAPK and MCP-1 was unregulated in the TAA group, with downregulation of Nrf2 was observed. TAA caused histopathological alterations associated with hepatic vacuolation and fibrosis, increasing collagen fibers and high immuno-expression of VEGF. On the other hand, treatment with BP successfully improved the severe effects of TAA on the liver and restored histological architecture. Our study concluded the protective potentials of BP for attenuating liver fibrosis and could be used as adjuvant therapy for treating hepatic fibrosis.

Lipopolysaccharide induced neuroprotective effects of bacterial protease against Alzheimer's disease in male Wistar albino rats.

Alzheimer's disease (AD) is a highly severe neurodegenerative condition that affects the hippocampus and is characterized by memory loss and dementia. This investigation aims to determine the potential of a bacterial protease enzyme produced by a new mutant strain of bacteria (Bacillus cereus S6-3/UM90) to influence the rat behavioural, biochemical, histological, and immuno-histochemical functions induced by lipopolysaccharides (LPS) experimentally. The administration of LPS exhibited a decline in memory performance via Morris' Water Maze test along with an elevation of IL-6, IL-17, amino acid neurotransmitters, Adenosine monophosphate (AMP), and 8-OHdG, whereas a decrease in ATP (Adenosine Triphosphate), monoamine transmitters, AChE (acetylcholinesterase) and PC (phosphatidylcholine). Additionally, there was a notable increase in GFAP (glial fibrillary acidic protein) and p-Tau protein immuno-expression levels along with obvious histological lesions in the hippocampal CA3 region. Moreover, the administration of protease or Donepezil restored the measured parameters to nearly normal levels and improved the histological architecture of the hippocampus and ameliorated memory impairments. In conclusion, the study provides evidence that the treatment with Bacterial protease can improve the memory and learning impairments of LPS-induced AD and may be used as a promising therapeutic agent to manage AD since it has anti-inflammatory and antioxidant effects.

Inflammatory Modulation of miR-155 Inhibits Doxorubicin-Induced Testicular Dysfunction via SIRT1/FOXO1 Pathway: Insight into the Role of Acacetin and Bacillus cereus Protease.

Doxorubicin (DOX) is a chemotherapeutic agent that can disrupt testicular function leading to male infertility. This study examined the protective role of natural flavone, acacetin (ACA), and a protease of Bacillus cereus bacteria (B. cereus) as well as the potential role of miR-155/SIRT1/FOXO1 network in DOX-induced testicular injury. Twenty-four male Wistar rats were randomly allocated into four groups and treated as follows: Control, DOX (1 mg/kg, i.p) every other day for 21 days with a total dose equal to 10 mg/kg throughout the experiment, and pre-treated groups that received ACA (5 mg/kg/day, p.o) or B. cereus protease (36 mg/kg/day, p.o) for a week prior to DOX administration. DOX challenge reduced the testis weight coefficient, serum testosterone, and testicular 17ß-hydroxysteroid dehydrogenase (17ß-HSD). DOX caused a significant increase in testicular oxidative stress, inflammatory, and apoptotic markers. Aberrant testicular miR-34c, a germ-specific miRNA, and miR-155 expressions were observed, along with decreased protein expression of sirtuin1 (SIRT1) dependent forkhead box 1 (FOXO1) acetylation which induces apoptosis. Besides, abnormal histopathological architecture and a marked reduction in the testicular expression of proliferating cell nuclear antigen (PCNA) were observed. ACA or protease administration significantly improved the histopathological and immunohistochemical pictures compared with DOX alone and renovated testicular functions. Interestingly, treatment with protease was more significant than treatment with ACA in ameliorating DOX-induced testicular injury. Taken together, this study reveals the prophylactic role of these two regimens on male fertility by exhibiting antioxidant, anti-inflammatory, and anti-apoptotic effects against DOX-elicited testicular damage, possibly via modulating miR-155/SIRT1/FOXO1 network.

Molecular characterization, catalytic, kinetic and thermodynamic properties of protease produced by a mutant of Bacillus cereus-S6-3.

The proteolytic strain Bacillus cereus-S6-3 was subjected to mutagenic treatments viz. UV irradiations and methyl methane sulfonate (MMS). The obtained mutant strain, B. cereus-S6-3/UM90 showed 1.34 fold over the parent strain. Molecular characterization of proteases from the parent (PP/S6-3) and mutant (PM/UM90) strains indicated that they were consisted of two domains and binds a zinc ion and 4 calcium ions in the active site. Amino acid sequence alignment of PM/UM90 protease showed 19 amino acid residues were substituted compared to that of the wild-type enzyme. However, both proteases contained equal number of aromatic and hydrophobic amino acids. Protease from PM/UM90 showed an effective improvement in thermal properties in terms of reaction temperature, t1/2, the values of kd, activation energy (Ea), and decimal reduction time (D) within the temperature range from 60 to 80 °C. In addition, the kinetic and thermodynamic parameters for substrate hydrolysis (i.e., Km, Vmax, ΔH*, ΔG*, ΔS*, kcat, Vmax/Km, kcat/Km, ΔG*E-T and ΔG*E-S) showed a significant improvement of the catalytic efficiency for PM/UM90 protease. Furthermore, the correlation between thermodynamic properties and the patterns of amino acid substitution of wild-type enzyme to has been discussed.

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1.

The present study demonstrates the production and thrombolytic potential of a novel thermostable thiol-dependent fibrinolytic protease by Bacillus cereus RSA1. Statistical optimization of different parameters was accomplished with Plackett-Burman design and validated further by central composite design with 30.75 U/mL protease production. Precipitation and chromatographic approaches resulted in 33.11% recovery with 2.32-fold purification. The molecular weight of fibrinolytic protease was 40 KDa and it exhibited a broad temperature and pH stability range of 20-80 °C and pH 5-10 with utmost activity at 50 °C and pH 8, respectively. The protease retained its fibrinolytic activity in organic solvents and enhanced the activity in solutions with divalent cations (Mn2+, Zn2+, and Cu2+). The enzyme kinetics revealed Km and Vmax values of 1.093 mg/mL and 52.39 µg/mL/min, respectively, indicating higher affinity of fibrinolytic activity towards fibrin. Also, complete inhibition of fibrinolytic activity with DFP and a 2-fold increase with DTT and ß-mercaptoethanol indicates its thiol-dependent serine protease nature. MALDI-TOF analysis showed 56% amino acid sequence homology with Subtilisin NAT OS = Bacillus subtilis subsp. natto. The fibrinolysis activity was compared with a commercial thrombolytic agent for its therapeutic applicability, and fibrinolytic protease was found highly significant with absolute blood clot dissolution within 4 h in in vitro conditions. The isolated fibrinolytic protease of Bacillus cereus RSA1 is novel and different from other known fibrinolytic proteases with high stability and efficacy, which might have wide medicinal and industrial application as a thrombolytic agent and in blood stain removal, respectively.