{"id":53992,"date":"2023-12-31T10:24:40","date_gmt":"2023-12-31T10:24:40","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=53992"},"modified":"2024-01-05T07:03:43","modified_gmt":"2024-01-05T07:03:43","slug":"electron-microscopic-studying-the-impact-of-ozone-on-chronic-hepatitis-c-patients-as-antifibrotic","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no4\/electron-microscopic-studying-the-impact-of-ozone-on-chronic-hepatitis-c-patients-as-antifibrotic\/","title":{"rendered":"Electron Microscopic Studying the Impact of Ozone on Chronic Hepatitis C Patients as Antifibrotic"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Hepatitis C virus is one of the greatest mutual reasons of chronic liver disease and a leading suggestion for liver transplantation. Because there isn’t a rodent model of enduring HCV infection, the pathophysiology of HCV-induced liver fibrosis is poorly known 1<\/sup>. Hepatocyte infection by HCV results in oxidative stress and the attraction of inflammatory cells after HCV avoids detection by the HLA-II-directed immune response. Both factors cause collagen formation and HSC activation. Additionally, a number of HCV proteins actively promote HSCs’ inflammatory and fibrogenic actions 2,3<\/sup>. Hepatitis C virus core protein causes mitochondrial damage, oxidative stress, and the expression of genes related to anti-oxidants.4<\/sup>. <\/p>\n\n\n\n

Low doses of ozone augment antioxidant endogenous\nsystems as catalase, superoxide dismutase and glutathione 5-6<\/sup>. After CCl4 poisoning, animals can maintain\nhepatocellular integrity by receiving ozone in atoxic dosages repeatedly. This\nallows for the induction of an adaptation to oxidative stress. 7<\/sup> . Ozone in a clinical histopathological study on 141\nchronic hepatitis C patients proved to have a potent antifibrotic and\nanti-inflammatory activity without affecting the virus itself 8 <\/sup>.<\/p>\n\n\n\n

Aim\nof the work<\/strong><\/p>\n\n\n\n

The objective of the study is to evaluate\nthe ultrastructural changes in liver biopsy sections compared with the\nhistopathologic results in chronic hepatitis C patients given ozone for 12\nweeks.<\/p>\n\n\n\n

Subjects and Procedures<\/strong><\/p>\n\n\n\n

Designing study<\/strong><\/p>\n\n\n\n

 
\n<\/em><\/strong>This study is a\nclinical phase II trial. Enrolled patients were given ozone\/oxygen gas mixture for\n12 weeks. Each patient gave their informed consent. The electron microscopy and\nhistopathology were performed in Theodor Bilharz Research Institute in Egypt.<\/p>\n\n\n\n

 Subjects\nSelection <\/strong><\/p>\n\n\n\n

 
\n<\/em><\/strong>Age 19\u201365 years was\nrequired for enrollment, irrespective to the presence of positive hepatitis C\nvirus blood indicators and sex for at least 6 months before enrollment in whom\ninterferon plus ribavirin treatment is not possible (Contra-indicated, failure\nof treatment or lack of compliance), unusual value of alanine transaminase\n(ALT) in serum within 6 months prior enrollment or evidence of hepatitis by\nultrasound (lymph nodes in porta-hepatis); negative hepatitis B virus (HBsAg)\nand; liver biopsy examination immediately showing the degree of hepatic\nfibrosis prior to enrollment from 0 to 6 according to Ishak modification of\nKnodell score, No previous history of administering the following medications:\nantiviral medications, immunoregulating medications, and other antifibrotic\nagents; a promise to refrain from receiving additional systemic antiviral\nmedications, cytotoxic medications, immunoregulators, medications that can\nlower serum enzyme activity and bilirubin levels, as well as herbal remedies including\nDDB, etc. The following scenarios should be avoided: individuals having\nHIV-positive testing results; elevated creatinine in serum 1.5 times higher\nthan normal; antinuclear antibody (ANA) titer more than a 1:160 dilution;\nindicative of autoimmune disorders.; uncompensated liver diseases; bone marrow\ninhibition; concurrence of other related diseases that might impact the current\ntreatment as unstable diabetes, pancreatitis, obvious manifestations of\nneurosis, alcoholic liver disease, disability of absorption, drug abuser,\npsychosis, malignant disease, unstable angina pectoris, renal insufficiency,\nand epilepsy, and so on; taking another medication in a clinical trial within\n30 days of the initial dose.<\/p>\n\n\n\n

Treatment methods and medications<\/strong><\/p>\n\n\n\n

     Afterwards\nselection and assessment, suitable candidates were given ozone\/oxygen gas\nmixture daily for 10 days in the form of major autohemotherapy (One hundred ml.\nozone\/oxygen gas mixture at a concentration of 25 \u03bcg\/ml. were mixed with 50 ml. blood aliquot) together\nwith 150 ml. ozone oxygen gas mixture by rectal insufflation in the same\nconcentration. Then the same treatment was given twice weekly for 12 weeks. Everyone\nunderwent follow-up once weekly during treatment and were followed up at\nout-patient department for 12 weeks after treatment.<\/p>\n\n\n\n

Assessment of blood and urine tests,\ntogether with relevant liver function indices \n<\/strong><\/p>\n\n\n\n

These were evaluated immediately before and\n12 weeks once the treatment has begun.<\/p>\n\n\n\n

Analysis of PCR for HCV RNA<\/strong>  <\/p>\n\n\n\n

Quantitative PCR was done immediately\nbefore treatment and 12 weeks from the start of treatment. <\/p>\n\n\n\n

Imaging examination (B mode ultrasound)<\/strong><\/p>\n\n\n\n

Abdominal ultrasound was done immediately\nbefore and after 12 weeks (end of treatment). The detection included the\nfollowing indexes: maximal oblique radius of right liver lobe, left lobe axis\nin middle line, main trunk diameter of portal vein, long axis of the spleen,\nliver surface, parenchymal echopattern and evidence of portal tract thickening\nfor associated shistosomiasis together with the presence or absence of lymph\nnodes in porta-hepatis. The ultrasound examination was carried out by fixed\noperators and on fixed equipment.<\/p>\n\n\n\n

Histopathological Assessment:<\/strong><\/p>\n\n\n\n

Needle liver biopsy specimens were obtained\nfrom all patients upon their written consent using trucut needle. Each specimen\nwas preserved in 10% buffered neutral formalin right away. delivered to the\nPathology Department where embed it into paraffin wax blocks, it was treated in\nvarious grades of alcohol. Paraffin sections (4mm thick) were cut; 3 from each specimen to\nbe stained with Haematoxylin & Eosin and Masson Trichrom stain for\nhistopathological evaluation and identification of the different grades of\ninflammatory activity and different stages of chronic hepatic lesions based on\nthe Ishak and Knodellscoring\nsystem 9-10<\/sup>. Histopathological samples were viewed and evaluated\nindependently. They were examined by a different pathologist who was not\ninvolved in the project from the start based on the observed results. The two pathologists’\nmutual consistency was satisfactory.<\/p>\n\n\n\n

Ultrastructure assessment by electron\nmicroscopy (EM):<\/strong><\/p>\n\n\n\n

Fifty two biopsies taken from 26 patients\nbefore and after treatment with ozone were subjected to (EM) examination.Liver\nbiopsies were prepared by fixing them in gluteraldehyde, washing them in equal\nparts of 0.3 M cacodylate and 0.4 M sucrose, postfixing them in 2% osmium\ntetroxide, dehydrating them in escalating alcohol concentrations, embedding\nthem in epoxy resin, and polymerizing them. Semithin liver sections stained\nwith methylene blue azur II were examined using light microscope for the\nidentification of the site for the performance of ultrathin sections. In this\nstudy, the midzonal area of the liver lobule was the site examined at the level\nof electron microscopy. Leica Ultracut R, an ultramicrotome, was used to create\nultrathin sections. They were inspected with a Philips EM 208 S and stained\nwith uranyl acetate and lead citrate. Examination of the following subcellular\ncriteria was performed: Signs of cellular regeneration and degeneration of\napoptotic changes, disorganized  lobular\narchitecture, changes in cellular organelles mitochondria, endoplasmic\nreticulum, free and attached ribosomes, lysosomes, peroxisomes, fat droplets,\nglycogen rosettes\u2026), nuclear changes, changes of bile canaliculi and\ncanaliuloductular junction, inflammatory cellular infilterate in the lobule of\nor circulating in blood sinusoids, presence or absence of kupffer cells,\nstellate cells,  presence or absence of\ncollagen in the Disse\u2019s space intercellularly or intracellularly.<\/p>\n\n\n\n

Therapeutic effects evaluation<\/strong><\/p>\n\n\n\n

This was based on Ishak modification of knoldell score of the liver biopsy. The patient was considered a responder to treatment when the grade of inflammation decreased 2 or more points and stage of hepatic fibrosis decreased at least 1 point at the end of the treatment course, compared with that before treatment.<\/p>\n\n\n\n

Indexes of non-invasive tests<\/strong> assessment  <\/strong><\/p>\n\n\n\n

In terms of clinical symptoms, blood liver\nfunction tests, and ultrasound detection data, these indices underwent thorough\nevaluation.<\/p>\n\n\n\n

Safety evaluation <\/strong><\/p>\n\n\n\n

The unusual clinical symptoms and results\nfrom any tests performed during treatment were noted.<\/p>\n\n\n\n

Statistical analysis<\/strong><\/p>\n\n\n\n

  Statistical\nanalysis was done via SPSS.9 software computer program. Results were expressed\nas means \u00b1 standard deviation of the means (SD). ANOVA test was used for\nmultiple group comparison. Data were summarized also using cross tabulation. p\nvalue = p< 0.05was used to determine statistical significance. <\/p>\n\n\n\n

Ethical consent<\/strong><\/p>\n\n\n\n

When the patient was still aware that the study would\nkeep his or her medical records private at all times, informed consent was\nobtained from the patient’s family members or from the patient himself. When\ncarrying out this research on humans, the World Medical Association’s\nDeclaration of Helsinki, its code of ethics, was adhered to. (FWA 00010609).<\/p>\n\n\n\n

Results <\/strong><\/p>\n\n\n\n

Selected patients<\/strong><\/p>\n\n\n\n

26 patients in all met the selection criteria, 11 females (42 %) and 15 males (58 %) (Table 1). All patients had a second liver biopsy at the end of treatment course (12 weeks). <\/p>\n\n\n\n

Table 1: Mean values of laboratory data of the study group (26 patients). <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/strong><\/p>\n<\/td>\n

\n

Before <\/strong><\/p>\n<\/td>\n

\n

After <\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Hemoglobin <\/strong>(g\/dl)<\/p>\n<\/td>\n

\n

13.64 \u00b1 1.58<\/p>\n<\/td>\n

\n

12.93 \u00b1 2.24<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

WBCs <\/strong>(109<\/sup>\/l)<\/p>\n<\/td>\n

\n

6.75 \u00b1 1.85<\/p>\n<\/td>\n

\n

6.24 \u00b1 2.0<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

RBCs <\/strong>(1012<\/sup>\/l)<\/p>\n<\/td>\n

\n

4.69 \u00b1 0.63<\/p>\n<\/td>\n

\n

4.84 \u00b1 0.80<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

HCt.<\/strong><\/p>\n<\/td>\n

\n

40.20 \u00b1 4.98<\/p>\n<\/td>\n

\n

36.0 \u00b1 9.25<\/p>\n<\/td>\n

\n

< 0.01<\/p>\n<\/td>\n<\/tr>\n

\n

Platelet count (106<\/sup>\/l)<\/strong><\/p>\n<\/td>\n

\n

243.79 \u00b1 70.45<\/p>\n<\/td>\n

\n

218.91 \u00b1 80.92<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

PT (sec.) <\/strong>(up to 11.7)<\/p>\n<\/td>\n

\n

12.31 \u00b1 0.92<\/p>\n<\/td>\n

\n

12.56 \u00b1 0.99<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

Prothrombin conc. (%) <\/strong>(80-100%)<\/p>\n<\/td>\n

\n

86.76 \u00b1 10.84<\/p>\n<\/td>\n

\n

82.66 \u00b1 13.92<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

PTT<\/strong><\/p>\n<\/td>\n

\n

33.77 \u00b1 3.68 (23)<\/p>\n<\/td>\n

\n

31.73 \u00b1 2.57<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

AST (U\/l) <\/strong>(5-38)<\/p>\n<\/td>\n

\n

52.0 \u00b1 31.74 (24)<\/p>\n<\/td>\n

\n

46.64 \u00b1 23.72<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

ALT (U\/l) <\/strong>(5-41)<\/p>\n<\/td>\n

\n

57.75 \u00b1 33.27<\/p>\n<\/td>\n

\n

38.64 \u00b1 29.78<\/p>\n<\/td>\n

\n

< 0.05<\/p>\n<\/td>\n<\/tr>\n

\n

T. bilirubin (mg\/dl) <\/strong>(up to 1.1)<\/p>\n<\/td>\n

\n

0.58 \u00b1 0.62<\/p>\n<\/td>\n

\n

0.45 \u00b1 0.13<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

Alk. Phosphatase <\/strong>(40-129)<\/p>\n<\/td>\n

\n

87.29 \u00b1 28.20 (24)<\/p>\n<\/td>\n

\n

62.64 \u00b1 15.94<\/p>\n<\/td>\n

\n

< 0.01<\/p>\n<\/td>\n<\/tr>\n

\n

Serum cholesterol<\/strong><\/p>\n<\/td>\n

\n

155.58 \u00b1 45.79<\/p>\n<\/td>\n

\n

142.82 \u00b1 31.74<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

Serum triglycerides<\/strong><\/p>\n<\/td>\n

\n

151.58 \u00b1 72.37<\/p>\n<\/td>\n

\n

128.36 \u00b1 37.64<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

HDL<\/strong><\/p>\n<\/td>\n

\n

43.10 \u00b1 15.01<\/p>\n<\/td>\n

\n

32.82 \u00b1 5.83<\/p>\n<\/td>\n

\n

< 0.01<\/p>\n<\/td>\n<\/tr>\n

\n

LDL<\/strong><\/p>\n<\/td>\n

\n

84.13 \u00b1 46.08<\/p>\n<\/td>\n

\n

87.82 \u00b1 33.42<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

Albumin (g\/dl) <\/strong>(3.5-5)<\/p>\n<\/td>\n

\n

4.18 \u00b1 0.44<\/p>\n<\/td>\n

\n

3.83 \u00b1 0.76<\/p>\n<\/td>\n

\n

< 0.05<\/p>\n<\/td>\n<\/tr>\n

\n

Total proteins (g\/dl) <\/strong>(6.4-8.2)<\/p>\n<\/td>\n

\n

7.83 \u00b1 1.66<\/p>\n<\/td>\n

\n

7.75 \u00b1 0.37<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

Globulin () ()<\/strong><\/p>\n<\/td>\n

\n

3.95 \u00b1 0.73<\/p>\n<\/td>\n

\n

3.92 \u00b1 0.95<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

A\/G ratio (%)<\/strong><\/p>\n<\/td>\n

\n

1.10 \u00b1 0.26<\/p>\n<\/td>\n

\n

1.04 \u00b1 0.31<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

Urea (mg\/dl) <\/strong>(10-50)<\/p>\n<\/td>\n

\n

26.92 \u00b1 8.63<\/p>\n<\/td>\n

\n

27.6 \u00b1 5.68<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

Serum creatinine ( up to 1.5 mg\/dl)<\/strong><\/p>\n<\/td>\n

\n

0.66 \u00b1 0.13<\/p>\n<\/td>\n

\n

0.75 \u00b1 0.15<\/p>\n<\/td>\n

\n

< 0.05<\/p>\n<\/td>\n<\/tr>\n

\n

Serum Uric acid (3.4-7.0 mg\/dl)<\/strong><\/p>\n<\/td>\n

\n

4.41 \u00b1 1.40<\/p>\n<\/td>\n

\n

4.6 \u00b1 1.71<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

INR<\/strong><\/p>\n<\/td>\n

\n

1.10 \u00b1 0.09<\/p>\n<\/td>\n

\n

1.14 \u00b1 0.12<\/p>\n<\/td>\n

\n

NS<\/p>\n<\/td>\n<\/tr>\n

\n

PCR<\/strong><\/p>\n<\/td>\n

\n

17059.25\u00b130370.31 (24)<\/p>\n<\/td>\n

\n

218736.18\u00b1545950.34 (17)<\/p>\n<\/td>\n

\n

< 0.05<\/p>\n<\/td>\n<\/tr>\n

\n

Grade of inflammation<\/strong><\/p>\n<\/td>\n

\n

10.33 \u00b1 3.32<\/p>\n<\/td>\n

\n

8.42 \u00b1 4.21<\/p>\n<\/td>\n

\n

< 0.001<\/p>\n<\/td>\n<\/tr>\n

\n

Stage of fibrosis<\/strong><\/p>\n<\/td>\n

\n

2.33 \u00b1 1.66<\/p>\n<\/td>\n

\n

2.0 \u00b1 1.67<\/p>\n<\/td>\n

\n

< 0.05<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Data were expressed as mean \u00b1 standard deviation.<\/p>\n

NS= not significant; p< 0.05= significant; p< 0.01= highly significant; p< 0.001=  very highly   significant.<\/p>\n\n\n

Analysis of observed indexes<\/strong><\/p>\n\n\n\n

Liver function<\/strong> (tab. 1, Fig 1)<\/strong>: <\/p>\n\n\n\n

The serum markers of inflammation (ALT, AST) and alkaline phosphatase (ALP) have improved after 12 weeks of treatment where the mean AST decreased from 52 to 46 (NS<\/em>) while the ALT and ALP showed more decrease, where ALT dropped from 58 to 39 (P<<\/em>0.05) and the ALP from 87 to 63 (P<<\/em>0.001). Changes in other parameters of liver function tests, coagulation profile, kidney functions and lipid profile were all within the normal range.<\/p>\n\n\n\n

Liver function tests (n=26)<\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: <\/strong>Mean values of liver function tests before and after treatment. <\/strong>*p < 0.05; **p< 0.001 relative to before treatment.<\/strong><\/p>\n

<\/p>\n

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Evaluation of results of PCR: Tab. (1) Fig. (2)<\/strong><\/p>\n\n\n\n

Follow up of viral kinetics by monitoring PCR count before and 12 weeks after the start of treatment showed increase of count, where after 12 weeks (end of treatment) the PCR raised from 17.059 to 218.736 (P<<\/em>0.01). <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: <\/strong>Mean PCR values before and after treatment. <\/strong>P< 0.01 relative to before treatment.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Liver histological examination: Tab (1), Fig. 3,4. <\/strong><\/p>\n\n\n\n

Evaluation of\nhepatic fibrosis based on Ishak modification of Knodell\u2019s score has shown\nmarked improvement in hepatic fibrosis and inflammatory activity. The mean\ngrade of inflammation dropped from 10.33 to 8.4 (P < 0.001) and the mean\nstage of fibrosis dropped from 2.33 to 2.0 (P value < 0.05). <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3:<\/strong> Grade of inflammation.<\/strong><\/p>\n

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Figure 4:<\/strong> Stage of fibrosis.<\/strong><\/p>\n

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Figure<\/strong> 5: Histopathological examination of liver sections  A: Section from patient  liver before treatment with ozone showing steatotic changes Activity score 12\/18 and stage fibrosis 3\/6  fibrosis (Haematoxylin and Eosin stain, X200).<\/strong><\/p>\n

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\"\"<\/td>\n\u0650Figure 6:<\/strong> Histopathological examination of liver sections A: Liver section from patient before treatment with ozone gas showing steatotic changes 16\/18 grade of inflammation and 4\/6 stage fibrosis (Haematoxylin and Eosin stain, X200).<\/strong>

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Results of electron microscopy (EM)<\/strong><\/p>\n\n\n\n\n\n
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Figure 7<\/strong><\/p>\n

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Figure 8<\/strong><\/p>\n

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Ultrastuctural manifestations of HCV infection was disclosed in liver specimens exposed or not to ozone treatment. HCV infection was characterized by the significant dilatation or hypertrophy of the cysternae of rough endoplasmic reticulum. These cysternae were laded with electron dense granular materials (Fig.7).<\/p>\n\n\n\n

Also, stage 4,5 and 6 liver samples the tubular configuration of the rough endoplasmic reticulum showed blind ballooned end filled with very fine electron dense proteinacious material or wrapping the mitochondria indicating that the cell was engaged in active protein synthesis. (Fig.8). Moreover electron dence C virus core proteins crowned the outer surface of the RER membrane especially in stage 5 and 6 (Fig 8).<\/p>\n\n\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 9<\/strong><\/p>\n

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Figure 10<\/strong><\/p>\n

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Virus like particles of about 30-40 nm in diameter were often disclosed in the examined sections (Fig.9).<\/p>\n\n\n\n

Condensation of cytoplasmic organelles around the nucleus was a common picture. The cytoplasm was occupied by electron dense granular proteinaciuous materials, polyribosomes, glycogen rosettes, and lipid deposits. Accumulated lipids appeared mainly as diffuse homogenous electron lucent deposits which look like hyaline material or it may take the form of circumscribed fat droplets (Fig. 10).<\/p>\n\n\n\n\n\n
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Figure 11<\/strong><\/p>\n

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Figure 12<\/strong><\/p>\n

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In cases classified at the level of light microscopy stage 4,5 and 6, wide intercellular spaces filled with homogenous electron lucent  materials with intervening intercellular microvilli was mainly seen (Fig. 11). <\/p>\n\n\n\n

Moreover bile canaliculi with stunted or lost microvilli was a common finding (Fig. 12).<\/p>\n\n\n\n\n\n
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Figure 13<\/strong><\/p>\n

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Figure 14<\/strong><\/p>\n

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Considerable glycogen deposits, collagen in the Disse\u2019s spaces, inter or intracellularly were detected in stage 5 and 6 (Fig. 13).<\/p>\n\n\n\n

On the other hand liver tissue exposed to ozone treatment revealed increase in peroxisomes and longitudinal orientation of mitochondrial cristae most probably engendered by increase exposure to oxygen (Fig. 13).<\/p>\n\n\n\n

Moreover, stellate cells with intracytoplasmic fat droplets are not seen in the examined liver sections taken from the patients after ozone exposure. Meanwhile, stellate cells were often encountered in unexposed samples to ozone especially in cases classified as stage 1 and 2. (Fig.14).<\/p>\n\n\n\n\n\n
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Figure 15<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Signs of cellular regeneration in the form of binucleated cells, RER enveloping the mitochondria, and hepatocyte progenitors seen insinuating between the cells at the sinusoid pole are seen (Fig. 15). Also apoptotic hepatocytes are rarely seen.Circulating inflammatory cells in the sinusoids and infiltrating the lobule were decreased in four cases after ozone exposure.<\/p>\n\n\n\n

Adverse effects<\/strong><\/p>\n\n\n\n

No clinically significant complication or side effect detected in a total number of settings exceeding 1000.<\/p>\n\n\n\n

Discussion <\/strong><\/p>\n\n\n\n

Several chronic liver disease patients have severe and ongoing liver damage led to hepatic fibrosis, a precursor to cirrhosis, which is characterized by an unusual accumulation of extracellular matrix. 11, 12, 13,14<\/sup>. Liver fibrosis denotes a chief universal healthcare burden. Current rehabilitation is inadequate to eliminate the causative cause. This approach has been effective in some diseases, mainly in chronic viral hepatitis and haemochromatosis 2,15,16,17<\/sup>. While other patients seeking medical attention were in an advanced stage of fibrosis, many patients’ conditions rendered treatment impossible 18,19<\/sup>. The lack of liver transplant donors has prompted researchers to look for broadly applicable antifibrotic treatments 20<\/sup>. <\/p>\n\n\n\n

Novel treatments for liver fibrosis are thus highly necessary. Over the past ten years, enormous advancements in our understanding of hepatic fibrosis have been made. Based on their capacity to undergo activation following liver injury from any cause, hepatic stellate cells play a crucial role 13, 21, 22, 23<\/sup>. It has been established that the majority of the excess extracellular matrix seen in chronic liver fibrosis is formed by hepatic stellate cells. The rational development of innovative antifibrotic medicines has been made possible by the thorough hepatic stellate cell biology knowledge 23<\/sup>. Based on the fundamental principles underlying the fibrogenic process, an effective treatment for hepatic fibrogenesis would likely also involve a number of other factors 24, 25, 26, 27<\/sup>.<\/p>\n\n\n\n

Antifibrotic drugs should work on several aspects of the formation and genesis of hepatic fibrosis because it is currently believed that treating antihepatic fibrosis and hepatic fibrosis are two dissimilar thoughts. Additionally, liver injury and the activation of hepatic stellate cells are caused by significant mechanisms such as oxygen stress and lipid peroxidation. Therefore, lipid peroxidation inhibition was an important strategy of antihepatic fibrosis 22, 24,<\/sup>. <\/p>\n\n\n\n

Oxidative stress is a key factor in liver\ndamage. ROS including superoxide anions, hydroxyethyl radicals, hydroxyl\nradicals and hydrogen peroxide are generated from a numerous of insults such as\nalcohol metabolism, ischemia\/reperfusion and drug\/toxin metabolites. ROS were implicated\nin apoptosis and necrosis of hepatocytes, and share in HSC activation 3, 8<\/sup>.\nNumerous major classes of free radical scavengers, as glutathione peroxidase\n(Gpx), catalase and superoxide dismutase (SOD), as well as SOD mimics, were\ninvestigated in many forms of liver injury, and they provided the tissue with\nefficient defense against oxidative damage 29,30<\/sup>. The therapeutic\nresponse achieved by ozone, after repeated administration is through the\noxidative stress induced in sub-injurious levels that can cause a\npreconditioning effect which is able to re-equilibrate the redox system changed\nby pathogenic stimuli. <\/p>\n\n\n\n

LOPs can function as physiological\nmessengers that can reactivate a disrupted biological system at submicromolar\nconcentrations 6<\/sup>. All\norgans can be affected by LOPs in trace amounts, especially the CNS and bone\nmarrow, notifying definite cell receptors of a calculated and minimal oxidative\nstress eliciting the adaptive response.<\/p>\n\n\n\n

In this study EM showed that liver tissue exposed to ozone treatment revealed increase in peroxisomes. On an average, human hepatocytes (liver cells) show ~1,000 peroxisomes. Half-life of peroxisomes is only 5 days. Enzymes for oxidation are generally found in peroxisomes. Their key enzymes are peroxidase and catalase; further they have superoxide-dismutase, uratoxidase, and D-amino acid oxidase. Inducing an adaptation to oxidative stress through repeated administration of ozone in atoxic dosages enables mice to maintain hepatocellular integrity after CCl4 poisoning. Low doses of ozone increased antioxidant endogenous systems such as catalase, superoxide dismutase and glutathione 5,6,9 <\/sup>.Ultra-structural manifestations of HCV infection was disclosed in liver specimens exposed or not to ozone treatment. This coincides with our results as we have disclosed that mean PCR increased with ozone treatment in a period of 12 weeks from 17.059 to 218.736. This also coincides with the results of Sadek 8<\/sup> where viral kinetics showed gradual increase of mean PCR after 10 and 20 days and 12 weeks. Circulating inflammatory cells in the sinusoids and infilterating the lobule were decreased in four cases after ozone exposure as seen on EM. This explains the significant decrease of liver enzymes in this study as well as in the study of Sadek 8<\/sup> where ALT, AST and ALK.Ph Showed marked improvement. The grade of inflammation also improved on histopathology from 10.33 to 8.42 by Ishak\u2019s score in this study compared to decrease from 10.08 to 7.94 in the study of Sadek 8<\/sup>.<\/p>\n\n\n\n

Stellate cells with intracytoplasmic fat droplets are not seen in the examined liver sections taken from the patients after ozone exposure on EM examination. Meanwhile stellate cells were often encountered in unexposed samples to ozone especially in cases classified as stage 1 and 2. Signs of cellular regeneration in the form of binucleated cells, RER enveloping mitochondria, hepatocyte progenitors seen insinuating between the cells at the sinusoid pole. Also apoptotic hepatocytes are rarely seen on the contrary of their common existence in specimens before treatment. Hepatic fibrogenesis and inflammatory reactions are triggered by the chronic hepatocellular mortality caused by necrosis and\/or apoptosis. Hepatocytes should ideally renew and replace dying cells; however, the imbalance of growth factors and the altered liver architecture and circulation frequently prevent hepatocellular regeneration in the case of chronic liver injury. Cellular apoptotic bodies and debris accumulate and cause inflammatory reactions, which may form a self-amplifying loop that further delay recovery from injury, and stimulates the fibrogenic process 31<\/sup>. In vitro, HSC might engulf the apoptotic bodies, and apoptotic bodies may also be phagocytosed by quiescent cells. Myofibroblast phenotypic transdifferentiation is made easier by HSC 32.<\/sup> . Hepatic biopsy samples from HCV infection and a rat model of bile-duct ligation both showed active HSC with engulfed apoptotic bodies. 33 <\/sup>. This explains the improvement of liver fibrosis after 12 weeks of ozone treatment where the mean stage of fibrosis dropped from 2.3 to 2.0 coinciding with the results of Sadek 8 <\/sup>where the mean stage of fibrosis dropped from 1.98 to 1.41 in a similar period of time of ozone treatment.  The improvement of liver inflammation and fibrosis scores despite elevation of the mean PCR count is similar to the effect of treatment of fibrosis using interleukin-10. Interleukin-10 medication for 12 months reduced intrahepatic inflammation and fibrosis in individuals with chronic HCV infection-mediated fibrosis who had not responded to antiviral therapy (mean change from 5 + 0.2 – 4.5 + 0.3 P\u20390.05) 21,34<\/sup>. During therapy, serum HCV RNA levels increased (mean HCV RNA at day 0, 12.3 + 3 mEq\/ml; at 12 months 38 mEq\/ml; P\u20390.05). <\/p>\n\n\n\n

Also\nthere was an obvious shift of lymphocyte response towards a Th2 predominant\nphenotype. Many lines of evidence seem to support the idea that HCV infection\nis non cytopathic, including histologic examination of liver biopsies and the\nfact that the many HCV carriers have high titer virus expression and have\nnormal liver enzymes and little or no damage in the liver 35,36,37,\n38,39,40,41<\/sup>. Ozone is well known to affect the immune system, and induces\nproduction of interleukin-10 in the body on treatment using the major\nautohemotherapy or rectal insufflation routes 40- 43<\/sup>. <\/p>\n\n\n\n

Conclusion\n<\/strong><\/p>\n\n\n\n

We\nconcluded that Ozone oxygen gas mixture is a direct anti-fibrotic and\nanti-inflammatory agent in treatment of chronic hepatitis C patients without\nimproving viral PCR as evidenced by histopathology, electron microscopy and\nquantitative PCR.<\/p>\n\n\n\n

Acknowledgment<\/strong><\/p>\n\n\n\n

We thanks all the\nparticipants.<\/p>\n\n\n\n

Conflicts of\nInterest <\/strong><\/p>\n\n\n\n

The authors\ndeclare that there is no conflict of interest.<\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

There is no funding Sources.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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    Introduction Hepatitis C virus is one of the greatest mutual […]<\/p>\n","protected":false},"author":15,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[111],"tags":[],"class_list":["post-53992","post","type-post","status-publish","format-standard","hentry","category-vol16no4"],"_links":{"self":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/53992","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/users\/15"}],"replies":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/comments?post=53992"}],"version-history":[{"count":5,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/53992\/revisions"}],"predecessor-version":[{"id":55159,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/53992\/revisions\/55159"}],"wp:attachment":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/media?parent=53992"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/categories?post=53992"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/tags?post=53992"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}