Anti-Fibrotic and Anti-Angiogenic Activities of Osbeckia octandra Leaf Extracts in Thioacetamide-Induced Experimental Liver Cirrhosis

Abstract

Chronic liver inflammation has become a major global health concern. In the absence of clinical surrogate markers to diagnose inflammatory liver disease, the intervention with effective drugs in modern medicine tends to be late. In Sri Lanka, traditional medical practitioners prescribe herbal preparations from Osbeckia octandra for the prevention and treatment of liver disorders. To test the efficacy of such treatments, we have administered thioacetamide (TAA) to male Wistar rats to induce chronic liver damage (disease control; DC) and examined how various leaf extracts: crude leaf suspension (CLS), boiled leaf extract (BLE), sonicated leaf extract (SLE), methanol leaf extract (MLE) and hexane leaf extract (HLE) of O. octandra ameliorate TAA-induced liver disease. The CLS, BLE and SLE treatments in cirrhotic rats significantly attenuated disease-related changes, such as liver weight and hepato-enzymes. The mRNA levels of Tnf-α were significantly decreased by 3.6, 10 and 3.9 times in CLS, BLE and SLE compared to DC. The same treatments resulted in significantly lower (19.5, 4.2 and 2.4 times) α-Sma levels compared to DC. In addition, Tgf-β1 and Vegf-R2 mRNA expressions were significantly lower with the treatments. Moreover, BLE expressed a strong anti-angiogenic effect. We conclude that CLS, BLE and SLE from O. octandra have potent hepatic anti-fibrotic effects in TAA-induced liver cirrhosis.

Keywords: Osbeckia octandra; Wistar rats; anti-angiogenic effect; cirrhosis; hepatoprotective; thioacetamide.

Figure 1

Aqueous extracts from O. octandra prevents body and liver weight retardation during the TAA challenge. (a) Experimental procedure. (b) Body weight gain from the beginning to the end from each of the seven groups of rats (HC, DC CLS, BLE, SLE, MLE or HLE treatment model) during the TAA challenge. The values are indicated in grams of increase/decrease using the starting weight. (c) Liver/body weight ratio at the end from each of the seven groups of rats. Error bars represent standard errors of the means (SEM) in each group (n = 5). The statistical significance has been indicated in two ways; (i) disease induction effect during the TAA challenge (HC vs. DC), (ii) O. octandra treatment consequence (DC vs. CLS, BLE, SLE, MLE and HLE), with one-way ANOVA followed by the Dunnett’s test. $, significant decrease; $, p = 0.05 to 0.01. *, significant increase; *, p = 0.05 to 0.01. ns, p > 0.05.

Figure 2

Selected aqueous extracts (CLS, BLE and SLE) protect the liver from TAA-induced damage by reducing the leakage of liver enzymes at the end of the treatment period. (a) Serum ALT levels, (b) serum AST levels and (c) serum ALP levels of HC, DC and after treatments of aqueous extracts along with TAA challenge. Error bars represent standard errors of the mean (SEM) in each group (n = 5). The statistical significance of results has been shown in two ways; (i) disease induction effect during the TAA challenge (HC vs. DC), (ii) O. octandra treatment effect (DC vs. CLS, BLE and SLE) was analyzed with one-way ANOVA followed by the Dunnett’s test. $, significant decrease; $, p = 0.05 to 0.01. *, significant increase; *, p = 0.05 to 0.01.

Figure 3

Aqueous extracts (CLS, BLE and SLE) demonstrated more potency to protect the liver against TAA challenge. (a) HC livers showed a normal appearance with a smooth surface. (b) Numerous nodules of various sizes can be seen on the liver surface of DC. (c) Livers treated with CLS showed a smooth surface. (d) Small nodules can be seen on the surfaces of livers treated with BLE and (e) SLE. (f,g) Nodules of variable sizes were present on the surfaces of livers treated with MLE and HLE. Bar = 10 mm.

Figure 4

Hepatoprotective effect of aqueous extracts (CLS and BLE) against TAA-induced liver cirrhosis. Histopathological sections of livers (stained with HE) from HC, DC and different leaf extract treatments of O. octandra CLS, BLE and SLE) after 15 weeks. (a) HC livers showed normal hepatic architecture and without histopathological changes. (b) Regenerating hepatic nodules formed by complete fibrous bridges were seen in the livers of DC. (c) Animals treated with CLS showed normal hepatic architecture. (d) Incomplete fibrous bridge formation was seen in the animals treated with BLE. (e–g) Complete fibrous bridge formation with regenerating hepatocytes was observed in animals treated with SLE, MLE and HLE, respectively. CV, central vein. Bar = 100 µm.

Figure 5

Aqueous extracts (CLS and BLE) showed an anti-fibrotic effect against TAA-induced liver cirrhosis. Histopathology of (stained with Masson’s trichrome) HC, DC and different leaves extracts of O. octandra treated CLS, BLE and SLE) livers at 15 weeks. (a) In the HC livers, collagen fibers were mainly seen in the vascular wall. (b) Increased collagen deposition in the thickened fibrous bridges was seen in DC. (c) A small amount of collagen was detected mainly in the periportal and centrilobular areas in livers treated with CLS. (d) Livers treated with BLE showed a few collagen deposits as thin fibrous bridges. (e–g) Increased amounts of collagen with thickened fibrous bridges were observed in livers treated with SLE, MLE and HLE. CV, central vein. Arrows indicate collagen depositions. Bar = 100 µm.

Figure 6

Treatments with aqueous extracts (CLS, BLE and SLE) showed a minimum amount of collagen deposits, indicating their hepatoprotective activity in TAA-induced liver cirrhosis. MT-stained liver sections were quantified using image J software. The values are indicated as percentages of the total surface. Error bars represent standard errors of the means (SEM) in each group (n = 5). The statistical significances are shown in two ways; (i) disease induction effect during the TAA challenge (HC vs. DC), (ii) O. octandra treatment consequence (DC vs. CLS, BLE, SLE, MLE and HLE), with one-way ANOVA followed by the Dunnett’s test. $, significant decrease; $$$, p < 0.001. *, significant increase; ***, p < 0.001. ns, p > 0.05.

Figure 7

mRNA expressions confirmed the establishment of liver cirrhosis in DC and the hepatoprotective effect of aqueous extracts in the other treatments. (a) Tnf-α, (b) α-Sma, (c) Tgf-β1 and (d) Vegf-R2 mRNA expressions determined by qPCR. Expression levels are normalized to 18S mRNA level. Error bars represent standard errors of the means (SEM) in each group (n = 5). The statistical significances are indicated in two ways; (i) disease induction effect during the TAA challenge (HC vs. DC), (ii) O. octandra treatment consequence (DC vs. CLS, BLE and SLE), with one-way ANOVA followed by the Dunnett’s test. $, significant decrease; $, p = 0.01 to 0.05, $$, p = 0.001 to 0.01, *, significant increase; **, p = 0.001 to 0.01. ns, p > 0.05.

Figure 8

Boiled leaf extract (BLE) of O. octandra inhibited angiogenesis in the HUVEC model. (a) Representative micrographs of 4 h post-seeding. (I) HUVECS were attached, aligned and organized in tubes with a lumen that appeared as a network when cultured on ECM. (II–IV) In the presence of O. octandra extract, HUVECs were attached but remained somewhat rounded as solitary cells. The effect of O. octandra was dose-dependent, and when the concentration increased from 250 to 1000 µg/mL, branching and tube formation was significantly inhibited. (b) Cumulative tube length and (c) number of branch points of capillary-like structures were also significantly reduced at 4 h. Error bars represent the standard error of the means (SEM) in each group (n = 3). The statistical significance was calculated with one-way ANOVA followed by Dunnett’s test. $, significant decrease; $$, p = 0.001 to 0.01.