Results were analyzed with GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA). The results (body weights, hematological parameters, serum biochemistry, and antioxidants) were recorded as mean ± standard error of mean (SEM). Data were subjected to one-way ANOVA and Tukey’s multiple comparison test at a significance value of 5%. Indices of fibrosity including AST/ALT, aspartate aminotransferase to platelet ratio index (APRI), and AST/PLT ratios were calculated.

Figure 1 shows the effect of treatment on the absolute and relative liver weights. CCl₄ and AWE treatment resulted in significant increases in absolute but not relative liver weight.

Table 1 shows the effect of treament on some hematological parameters of animals. Treatment had no significant effect on the haematological parameters, except for a decrease in platelet (PLT) levels in CCl₄-only treated group (p < 0.05). AWE and Silymarin treatment restored PLT levels to near normal.

Table 2 shows the effect of treatment on the biochemical and antioxidant profiles of animals. CCl₄ treatment resulted in significant increases in ALT, AST, ALP and bilirubin levels. Silymarin and AWE treatment resulted in decreases in these functions to near-normal levels. No significant changes were observed for the pro- and anti-oxidant status of the liver, except for an increase in the levels of reduced GSH in Silymarin co-treated group.

Figure 1

Table 1

Parameters	Normal	CCl4 only	CCl4 + Sily	CCl4 + AWE
WBC (x 1000/µL)	10.30 ± 1.04	12.30 ± 1.55	8.48 ± 0.62	9.10±0.79
RBC (x 106/µL)	8.61 ± 0.19	8.96 ± 0.14	8.62 ± 0.21	8.56±0.23
HGB (g/dL)	14.60 ± 0.26	15.78 ± 0.23	14.58 ± 0.34	14.75±0.15
HCT (%)	51.27 ± 0.64	54.43 ± 1.27	51.10 ± 1.28	51.85±1.19
MCV (fL)	59.53 ± 0.73	60.75 ± 0.70	59.33 ± 1.09	60.60±0.58
MCH (pg)	16.93 ± 0.23	17.60 ± 0.10	16.93 ± 0.19	17.28±0.43
MCHC (g/dL)	28.47 ± 0.18	29.00 ± 0.28	28.50 ± 0.19	28.48±0.54
PLT (x 103/µL)	887.67 ± 34.17	732.50 ± 58.35a	884.00 ± 30.48b	832.50±72.66b
LYM (%)	78.50 ± 6.26	73.43 ± 5.78	72.35 ± 1.52	64.28±7.14
NEUT (%)	21.50 ± 6.26	26.58 ± 5.78	27.65 ± 1.52	32.75±6.55
LYM (x 103/µL)	8.17 ± 1.37	9.13 ± 1.55	6.15 ± 0.51	5.80±0.71
NEUT (x 103/µL)	2.13 ± 0.62	3.18 ± 0.56	2.33 ± 0.16	3.08±0.75
RDW-SD (fL)	31.37 ± 0.44	32.00 ± 0.67	31.30 ± 0.47	32.58±0.71
RDW-CV (%)	13.00 ± 0.44	13.00 ± 0.30	13.33 ± 0.50	14.65±0.42
PDW (fL)	7.33 ± 0.03	7.95 ± 0.23	7.85 ± 0.13	8.25±0.73
MPV (fL)	6.53 ± 0.03	6.95 ± 0.13	6.80 ± 0.07	7.10±0.40
P-LCR (%)	4.30 ± 0.32	5.95 ± 0.51	5.35 ± 0.27	7.35±2.46
PCT (%)	0.58 ± 0.02	0.62 ± 0.03	0.50 ± 0.03	0.59±0.05

Statistical difference: a: different from Normal at p < 0.05 – 0.001; b: different from CCl₄ only at p < 0.05 – 0.001.

Table 3 shows the effect of the treatment on some non-invasive indices of liver fibrosis. CCl₄ resulted in significant increases in AFP and AST/PLT ratio and decreases in AST/ALT ratio and APRI. Silymarin and AWE prevented fibrosis based on observations with AFP. APRI was significantly lower in the Silymarin group compared with CCl₄-only group, while the AST/PLT ratio was reduced in the AWE group.

Table 3

	Normal	CCl4	CCl4 + Sily	CCl4 + AWE
AFP (ng/mL)	3.85 ± 0.15	5.29 ± 0.07a	4.72 ± 0.33	4.17 ± 0.32
AST/ALT	3.67 ± 0.64	0.83 ± 0.07a	2.27 ± 0.36ab	0.99 ± 0.06a
APRI (%)	0.09 ± 0.01	0.13 ± 0.01a	0.076 ± 0.01b	0.08 ± 0.01b
AST/PLT Ratio	44.86 ± 4.31	64.63 ± 6.35a	45.82 ± 5.64b	40.69 ± 6.12b

Statistical difference: a: different from Normal at p < 0.05 – 0.001; b: different from CCl₄ only at p < 0.05 – 0.001.

Figure 2(A-D) shows representative liver sections of normal and treated groups. CCl₄ treatment resulted in severe hepatocellular vacuolar degenerations with extensive coagulation necrosis of hepatocytes, inflammation, and fibroblast proliferation (Figure 2B). Silymarin and AWE treatment reversed these injuries to near normal architecture, especially as it relates to the necrosis of hepatocytes, as there were a few regenerative hepatocytes.

Figure 2

Figure 3 shows the micrograph of liver sections of normal and treated groups for the expression of interleukin (IL)-17 and 23, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and cyclooxygenase-1 (COX-1). Figure 4 shows the percent intensity of the expression of the cytokines. CCl₄ treatment resulted in the overexpression of these cytokines while AWE treatment suppressed these to near-normal levels.

Figure 3

Figure 4

Liver damage is associated with a significant increase in the serum levels of some enzymes which are normally segregated in the parenchymal cells of the liver. Examples of these enzymes include AST, ALP and ALT3. CCl₄ administration increased the serum levels of the aforementioned enzymes, indicative of liver damage. CCl₄, an intrinsic liver toxin, is known to mediate its toxic activity by the generation of free radicals and induction of oxidative stress5. Once metabolized, CCl₄ results in primary and secondary bond formation (a covalent bond formation with DNA, proteins, lipids, and carbohydrates), which results in cell damage. Damage to the hepatocytes then leads to the leakage of these enzymes into the serum6. Previous studies revealed the presence of flavonoids and hydrolysable tannins in the hydroethanolic extracts of A. wilkesiana, and these possess high antioxidant activity8. This explains the observed decrease in the ALT, AST and ALP levels upon administration of CCl₄ and AWE. In the case of AST and ALP, the levels were near normal.

Consistent with results from a previous study, administration of 1 ml/kg b.wt. CCl₄ intraperitoneally for 8 weeks resulted in a marked increase in the serum bilirubin levels (direct and indirect) and a decrease in total cholesterol and triglyceride levels23. This is indicative of liver damage. However, administration of plant extract did not have any significant effect on the serum cholesterol and triglyceride levels.

There is a common association between hepatic fibrosis and oxidative stress. Free radicals result in peroxidation of membrane lipids, thereby leading to cell damage. Thus, antioxidants may help in reducing the progression of liver fibrosis24. Results from this study showed no significant effects of CCl₄ on MDA levels; however, there was some decrease in the GSH levels. Decreased GSH levels upon CCl₄ administration was also observed in the previous studies25. High antioxidant activity evident by high concentrations of flavonoids and tannins in the hydroethanolic extract of A. wilkesiana8 explains the increase in GSH levels in the CCl₄+AWE group. The increase was, however, very prominent in the Silymarin-treated group, confirming the high degree of hepatoprotection.

Biopsy of the liver remains the ultimate technique for assessing hepatopathies26, such as for example, detecting and determining the stage of fibrosis in non-alcoholic steatohepatitis27. The procedure is invasive, however, and has risks of serious complications. Other cost-effective and useful means that are easy to measure and handle include platelet count, AST/ALT ratio, AST-to-platelet ratio index (or APRI), etc.26 An increase in the APRI was seen in the CCl₄ control group. Administration of Silymarin and AWE resulted in a decrease in the APRI. Blood platelet levels and AST levels are known to decrease and increase respectively with fibrosis progression26. Thus, an increase in APRI is shown to be an index of the severity of hepatic disease28, even in animal models. Though the APRI alone is likely not sufficiently sensitive to rule out significant disease, the lower the APRI score (less than 0.5), the greater the negative predictive value (and ability to rule out cirrhosis); the higher the value (greater than 1.5), the greater the positive predictive value (and ability to rule in cirrhosis) 29. CCl₄ was, therefore, confirmed to have caused hepatotoxicity, which was reversed in the AWE and Silymarin-treated groups.

The current study showed that Silymarin and AWE improved PLT levels compared with CCl₄-only group. Cirrhotic rats induced with dimethylnitrosamine and 70% hepatectomy were demonstrated to have improved platelet levels by a single intravenous injection of thrombopoietin which correlated with the inhibition of HSC activation and decrease of the fibrotic area in the liver, while antiplatelet serum attenuated hepatic regeneration30. It has been suggested that platelets may promote hepatocyte proliferation by secreting hepatocyte growth factor (HGF), a potent mitogen for hepatocytes. HGF is known to activate MET receptor essential for organogenesis and wound healing. Further, HGF may contribute to the resolution of fibrosis by modulating levels of TGF-β and matrix metalloproteinases (MMPs), which are the main ECM enzymes degrading collagen31. Platelets are known to induce hepatic regeneration by directly interacting with hepatocytes by promoting cell cycle progression and metabolic pathways in hepatocytes32.

Furthermore, as mentioned earlier, AWE and Silymarin treatments restored the histology of the liver after CCl₄ damage to near normal architecture. Also, the high expression of IL-17, IL-23, NF-κB and COX-2 in the CCl₄ treated group shows the necrotic and inflammatory reactions in CCl₄-induced toxicity. NF-κB and COX-1 as pro-inflammatory signals and their roles in liver fibrosis have been well established33, 34. IL-17 has been shown to directly promote the production of IL-6 and IL-8 in human dermal fibroblasts35 with in vitro studies showing an increased proliferation of fibroblasts in vascular endothelial cells, suggesting the pivotal role of IL-17 in fibrosis and endothelial inflammation36, 37. IL-17 is a major pro-inflammatory cytokine involved in neutrophil recruitment38. IL-23 is a pro-inflammatory heterodimeric cytokine composed of an IL12B (IL-12p40) subunit (that is shared with IL12) and the IL23A (IL-23p19) subunit39. In psoriasis, IL-23 expression is markedly upregulated, compared to normal skin40, and also in chronic bowel inflammation41. Thus both IL-17 and 23 are significantly involved in inflammation, leading to fibrosis. In the current study, AWE was observed to significantly downregulate the expression of these pro-inflammatory molecules, underscoring the healing properties of the extract.

It can be suggested that the subchronic protective effect of A. wilkesiana ‘inferno’ leaf extracts on the liver of animals against CCl₄-induced toxicty is by protecting tissues membranes from peroxidation, leakage of enzymes, and improvement of the microstructure. Also, the extracts protect against the overexpression of pro-inflammatory cytokines and related activities leading to cell death.

This study indicates that Acalypha wilkesiana ‘inferno’ possesses subacute protection against CCl₄-induced toxicity by reducing the levels of ALT, ALP and AST, as well as AFP, AST/PLT ratio and APRI. The protective effect of this extract was supported by histological and immunohistochemistry observations. Thus, 50% hydroethanolic extract of A. wilkesiana inferno can be developed as an effective hepatoprotective agent which brings about the functional improvement of liver function and can be exploited as a therapeutic agent against liver fibrosis.

α-SMA: alpha-smooth muscle actin

AFP: alpha-fetoprotein

ALP: alkaline phosphatase

ALT: alanine aminotransferase

ALW: absolute liver weight

APRI: aspartate aminotransferase to platelet ratio index

AST: aspartate aminotransferase

AWE: Acalypha wilkesiana hydroethanolic extract

CCl₄: Carbon tetrachloride

COX-1: clyclooxygenase-1

DBil: direct bilirubin

DNA: deoxyribonucleic acid

GPx: glutathione peroxidase

GSH: reduced glutathione

GST: glutathione transferase

HCT: haematocrit

HDL: high density lipoproteins

HGB: haemoglobin concentration

HSC: hepatic stellate cells

IBil: indirect bilirubin

IL-17: interleukin-17

IL-23: interleukin-23

LYM: lymphocytes

MCH: mean corpuscular haemoglobin

MCHC: mean corpuscular haemoglobin concentration

MCV: mean corpuscular volume

MDA: malondialdehyde

mRNA: messenger ribonucleic

Nfkβ: nuclear factor kappa B

PCT: Plateletcrit

PDW: platelet distribution width

P-LCR: platelet larger cell ratio

PLT: platelet

RBC: red blood cell

RDW: red cell distribution width

RLW: relative liver weight

SOD: superoxide dismutase

TBil: total bilirubin

TChol: total cholesterol

TGF-β: tumour growth factor-β

Trig: triglycerides

VLDL: very low density lipoproteins

WBC: white blood cell

WHO: world health organization

Not applicable.

C. Larbie conceived and designed the experiments; analyzed and interpreted the biochemical and hematological data; BO Emikpe and T.A. Jarikre contributed reagents and performed all histology and immunohistochemical experiments; A.A. Oyagbemi contributed reagents and performed all liver antioxidant experiments; C. Larbie, C. Owusu Adjei, R.A. Nyarko and E.B. Aseidu managed all literature search and drafted the manuscript. All authors approved the final manuscript.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasionable request.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.