Tumor cell death visualization of renal cell carcinoma under the combined effect of the Gratiola officinalis extract and cyclophosphamide using fluorescent staining methods

Objective of the study:We used fluorescence imaging methods of apoptosis and necrosis in human renal carcinoma A498 tumor cells in vitro to reveal the indicated forms of cell death under the combined effect of flavonoid-containing extract of Gratiola officinalis and cytostatic(cyclo-phosphamide).Materials and methods:The dyes were propidium iodide and acridine orange,which were used in the"alive and dead"test.This test helped us to identify the total number of dead cells in the forms of necrosis and apoptosis and the number of cells in which apoptosis had started,it was characterized by the appearance of apoptotic bodies or nucleus pyknosis.Results:We found the most pronounced cytotoxic activity at the ratio of extract of Gratiola officinalis and cyclophosphamide concentrations of 1∶1.The number of living cells decreased when exposed to the ratio of extract and cytostatic concentrations of 2∶1.When the ratio of concentration of the extract relative to the cytostatic increased to 3∶1,the cytostatic activity of the extract began to appear,the total number of tumor cells decreased.The number of cells with nucleus pyknosis and the number of cells with apoptosis signs significantly increased at a 3∶1 ratio of extract and cytostatic concentrations,which confirms the presence of pro-apoptotic activity of the studied combination.This trend indicates the dependence of a certain form of cell death(apoptosis,necrosis)on the ratio of extract and cytostatic doses,and it also demon-strates the cytostatic and cytotoxic effects of this combination.Conclusion:Fluorescence methods of investigation in the"alive and dead"test allowed us to visualize the forms of cell death of human kidney carcinoma A498 by combined exposure to the fiavonoid-containing extract of Gratiola officinalis and cytostatic(cyclophosphamide)24 h after exposure.We found that the combination with a concentration ratio of the extract and cyclophosphamide of 3:1 has the greatest effectiveness due to stimulation of the cytostatic effect and cytotoxic effect.

Research progress on fluorescence analysis of antitumor Chinese medicine components

This paper classifies the anti-tumor traditional Chinese medicine's fluorescence analysis and finds that its research content mainly includes two aspects:the application of fluorescence analysis in the determination of the effective component content of anti-tumor traditional Chinese medicine;the study of the combined use of fluorescence imaging technology and the drug.According to literature analysis,it is found that although the research on the fluorescence analysis of anti-tumor Chinese medicine components is in a good state of development,it still needs to explore further in the identification of unknown components and deeper research on known components.Therefore,it can provide a certain basis for clinical guidance of tumor patients.

Current Problems of the Diagnostics and Treatment of Sepsis and Burn Injuries: The Modified Pathogenetic Concept

Background: The deep understanding of pathogenesis is a key moment in the formation of the modern strategy of modern medicine. We conducted the thorough analysis of the microscopic processes occurring in the bodies of patients with purulent-septic complications. The modified pathogenetic concept of the diagnostic and treatment model of diseases with septic complications is presented. The obtained information about the mechanisms of origin and development of these diseases is fundamentally important for finding the modern effective methods of treating patients. The aim of the research is to modify treatment tactics for patients with sepsis and burn injuries based on the modified pathogenetic concept using modern diagnostics, i.e. the method of fluorescence spectroscopy (MFS) and biomarkers. Materials and Methods: The proposed modified pathogenetic concept of the diagnostic and treatment model of diseases with purulent-septic complications along with standard methods was used successfully for effective treatment of 15 patients with sepsis and 25 with burn injuries. Results: 3 main scenarios of behaviour of spectral-fluorescence characteristics of patients with sepsis are illustrated. Spectral-fluorescence markers of sepsis were studied, which are informative 24 to 48 hours before the appearance of obvious clinical and laboratory signs of significant changes in the general somatic status of patients. Conclusions: The proposed diagnostic and therapeutic approach is new and fundamentally important for diagnostics and monitoring of the process of treatment of patients with purulent-septic diseases and burn injuries. An in-depth understanding of the dynamics of septic complications and the corresponding changes of the main markers of these diseases during treatment is especially relevant. The use of infusion therapy with solutions of donor albumin as an effective pathogenetic treatment is scientifically justified.

Current Problems of Diagnostics and Treatment of Purulent-Inflammatory Diseases and Sepsis in Medical Practice

Background: Poor treatment of burns can lead to sepsis and even death. Especially promising is the use of the method of fluorescence spectroscopy for its diagnostics. The aim of the research is to create a pathogenetic concept as the basis of a diagnostic and treatment model of purulent-inflammatory diseases and sepsis. Material and methods: The study lasted from 2001 to 2019 and included 4 stages. The experimental base of luminescent research at all stages was the luminescence laboratory of the Department of Experimental Physics of the Ivan Franko National University of Lviv. The study was performed by using optical monochromators MDR-2 and MDR-12. The excitation of the blood serum was performed with light with a wavelength of 280 nm, which corresponds to the glow region of human serum albumin. Results: The main indicators, used for the analysis, were the fluorescence intensity (IF) and the position of the maximum fluorescence band (λmax). The object of the study was samples of the blood serum of patients of the main and control groups. In the case of the presence of endogenous intoxication in the body, albumin binding centers are blocked by the products of bacterial metabolism and therefore such “pathological” albumin is unable to perform its functions, including transport and detoxification. Conclusions: The pathogenetic concept as the basis of the diagnostic and treating model of purulent-inflammatory diseases and sepsis was devised. In particular, the important role of albumin solution infusion in the treatment of the above-mentioned diseases was established.

The Pathogenetic Concept of the Diagnostic-Treatment Approach for Patients with Purulent-Septic Complications

Background: The significant improvement of immediate and long-term functional results of treating patients is the fundamental problem of modern medical science. A deep understanding of the pathogenesis is the key point in creating the management strategy for patients with various diseases. Information about the mechanisms of origin and development of purulent-inflammatory diseases and sepsis is essential for finding effective ways to prevent and treat them. The aim of the research is to use the method of fluorescence spectroscopy in creating the pathogenetic diagnostic and treatment model for the prevention and treatment of purulent-inflammatory diseases and sepsis, modification of treatment tactics, search for new markers of purulent-septic diseases, as well as monitoring of patients during the treatment. Materials and methods: The proposed approach, along with standard diagnostic methods, was used to organize the treatment process of 100 patients with purulent-inflammatory diseases, including 15 patients with sepsis, 35 with acute inflammatory abdominal pathology, 20 patients with burn injury (main group) and 35 patients with burn injury (comparison group). Results: The behavior of spectral-fluorescent characteristics in their dynamics has been studied, and the new markers for assessing patients’ conditions have been proposed. Their effectiveness for the diagnosis of purulent-septic diseases has been proved, which advances the results of standard research methods by 24 - 48 hours. Conclusions: The proposed diagnostic and treatment approach is fundamentally important for diagnosis and monitoring during the treatment of patients with purulent-septic diseases. Particularly relevant is the proposal to modify the treatment process for these patients, associated with the use of infusion of donor albumin solutions.

The New Approach to the Diagnostics and Treatment of Endogenous Intoxication in Patients with Burn Injury

Background: The main causes of mortality in patients with burn injury are the development of systemic inflammatory process, multiple organ failure and septic complications. The aim of the research: Improvement of diagnostic and therapeutic approaches to the detection and elimination of endogenous intoxication in patients with burn injury. Materials and methods: The main study group consisted of 19 patients and the comparison group—of 10 patients with burn injury. 92 blood serum (BS) samples of the patients of the main group were tested, using the method of fluorescence spectroscopy (MFS). The advanced therapeutic tactics were proposed for the patients of both groups. The control group consisted of 40 healthy individuals (donors). BS of these patients was also tested using MFS. Results: Patients with burn injury have endogenous intoxication in their blood. The effective concentration of albumin is reduced in patients with burn injury due to the blockage of albumin binding centers by bacterial metabolism products. Fluorescence spectra (FS) of BS in patients with burn trauma and donors were obtained and investigated. Based on MFS results, an improved treatment regimen using infusion of albumin solution was proposed. Conclusions: An improved technique for the management of patients with burn injury is based on the use of MFS for the diagnostic evaluation of endogenous intoxication in them. The idea of pathological changes in albumin molecules in patients with burn injury is pathogenetically substantiated by the successful use of infusion of albumin solution in these patients on the basis of the MFS.

New approaches to diagnostics and treatment of cholangiocellular cancer based on photonics methods

Cholangiocellular cancer(CCC)is an oncological disease of the bile ducts characterized by a high mortality rate.To date,the use of standard methods for the diagnosis and treatment of CCC has not been able to reduce mortality from this disease.This work presents the results of fluorescence diagnostics(FD),which consists in using a modified optical fiber and photodynamic therapy(PDT)using a therapeutic laser instead of a low-intensity laser.This technique was tested on 43 patients in a clinical setting.The results obtained indicate a direct correlation between spectroscopic and video FD methods.Furthermore,a direct correlation was found between the photobleaching of a chlorin e6-based photosensitizer,with the commercial names of PhotoIon Radachlorin and Photoran and stricture regression.Our findings demonstrate the possibility of using a therapeutic laser with a wavelength of 660 nm for both diagnosis and treatment of bile ducts cancer,which results in a significant reduction of the operation time without decreasing its effectiveness.

Study of possibility of cell recognition in brain tumors

The brain has an exceptionally high requirement for energy metabolism,with glucose serving as the exclusive energy source.Cancers,including glioblastoma,have a high glucose uptake and rely on aerobic glycolysis for energy metabolism.The alternation of high-efficiency oxidative phosphorylation to a low-efficiency aerobic glycolysis pathway(Warburg effect)provides macromolecules for biosynthesis and proliferation.Current research indicates that the specific metabolism in the tumor tissue and nonnal brain tissue in the glioma allows the use of 5-aminolevulinic acid(5 ALA)-induced protoporphyrin IX(PpIX)and methylene blue(MB)to monitor and correct the development of the tumor.The focus is on the detection of the differences between tumor cells and tumorassociated macrophages/microglia using spectroscopic and microscopic methods,based on the fluorescent signals and the difference in the drug accumulation of photosensitizers(PSs).Since 5 ALA has long been used effectively in the clinic for fluorescent surgical navigation,it was employed as an agent to identify the localization of tumor tissue and study its composition,particularly tumor and immune cells(macrophages),which have also been shown to actively accumulate PpIX.However,since PpIX is photodynamically active,it can be considered effective as the main target of tumor tissue for further successful photodynamic therapy.MB was employed to visualize resident microglia,which is important for their activation/deactivation to prevent the reprogramming of the immune cells by the tumor.Thus,using two drugs,it is possible to prevent crosstalk between tumor cells and the immune cells of different geneses.